Fibroblast growth factor homologous factor-4

ABSTRACT

The invention provides fibroblast growth factor homologous factor (FHF) polypeptides and nucleic acid molecules that encode them. Also included in the invention are diagnostic and therapeutic methods using FHF polypeptides and nucleic acids.

This application is a divisional of U.S. Ser. No. 08/705,245, filed Aug.30, 1996 (now U.S. Pat. No. 6,020,189).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to polypeptide growth factors andspecifically to fibroblast growth factor homologous factors (FHFs) andnucleic acids encoding FHFs.

2. Description of Related Art

The fibroblast growth factor (FGF) family encompasses a group ofstructurally related proteins with a wide range of growth promoting,survival, and differentiation activities in vivo and in vitro (reviewedin Baird and Gospodarowicz, N.Y. Acad. Sci., 638:1, 1991; Eckenstein, J.Neurobiology, 25:1467, 1994; Mason, Cell, 78:547, 1994). As of June,1996, nine members of this family had been characterized by molecularcloning and their sequences published. The first two members of thefamily to be characterized, acidic FGF (aFGF/FGF-1) and basic FGF(bFGF/FGF-2), have been found in numerous tissues, including brain, eye,kidney, placenta, and adrenal tissues (Jaye, et al., Science, 233:541,1986; Abraham, et al., Science, 233:545, 1986). These factors have beenshown to be potent mitogens and survival factors for a variety ofmesoderm and neuroectoderm-derived tissues, including fibroblasts,endothelial cells, hippocampal and cerebral cortical neurons, andastroglia (Burgess and Maciag, Ann. Rev. Biochemistry, 58:575, 1989).Another member of the FGF family is int-2/FGF-3, which is encoded by agene that is a common target for activation by the mouse mammary tumorvirus, and therefore is presumed to be an oncogenic factor (Smith, etal, EMBO J., 7:1013, 1988). The genes encoding FGF-4, FGF-5, and FGF-6have transforming activity when introduced into NIH 3T3 cells(Delli-Bovi, et al, Cell, 50:729, 1987; Zhan, et al., Mol. Cell. Biol.,8:3487, 1988; Marics, et al., Oncogene, 4:335, 1989), while keratinocytegrowth factor (KGF)/FGF-7, FGF-8, and FGF-9 are mitogenic forkeratinocytes, mammary carcinoma cells, and astrocytes, respectively(Finch, et al, Science, 245:752, 1989; Tanaka, et al, Proc. Natl. Acad.Sci. USA, 89:8928, 1992; Miyamoto, et al., Mol. Cell Biol., 13:4251,1993). Recent experiments indicate that several FGFs have bioactivitiesthat were not evident during their initial identification. For example,FGF-2 has been shown to induce ventral mesoderm in Xenopus embryos(Slack, et al., Nature 326:197-200, 1987; Kimmelman, et al., Cell51:869-877, 1989), FGF-4 has been shown to be involved in growth andpatterning of the chick limb bud (Niswander, et al., Nature 371:609-612,1994), FGF-5 has been shown to control hair follicle cycling in themouse (Hebert, Cell 78:1017-1025, 1994), and FGF-8 has been shown tocause duplications of the embryonic chick midbrain (Crossley, et al.,Nature 38:66-68, 1996). Several of the FGFs, including aFGF (FGF-1) andbFGF (FGF-2), lack classical signal sequences, and the mechanism bywhich they are secreted is not known. Current data indicate that FGF-1and FGF-2 are released from cells by a route that is distinct from theER-Golgi secretory pathway (Florkiewicz, et al., J. Cell Physiol.162:388-399, 1995; Jackson, et al., J. Biol. Chem. 270:33-36, 1995).

The nine published members of the FGF family, FGFs 1-9, are between 155and 268 amino acids in length and share approximately 25% or more aminoacid sequence identity, as well as a conserved central region ofapproximately 140 amino acids. This region forms a compact beta-barrelwith three-fold symmetry that is nearly identical in structure to thefolded core of interleukins 1-alpha and 1-beta (Zhu, et al., Science251:90-93, 1991; Zhang, et al., Proc. Natl. Acad. Sci. USA88:3446-3450,1991; Eriksson, et al., Proc. Natl. Acad. Sci. USA88:3441-3445, 1991; Ago, et al., J. Biochem. 110:360-363, 1991). FGF-1and FGF-2 also resemble interleukin 1-beta in lacking a classical signalsequence.

FGF signaling is generally thought to occur by activation oftransmembrane tyrosine kinase receptors. For example, FGF-1, FGF-2, andFGF-7/KGF have been shown to exert some or all of their biologicalactivities through high affinity binding to such receptors (see, e.g.,Lee, et al., Science, 245:57, 1989; reviewed in Johnson and Williams,Adv. Cancer Res., 60:1, 1993). Four FGF receptor (FGFR) genes have beenidentified thus far (Johnson, et al., Adv. Cancer Res. 60:1-41, 1993),and activating or inactivating receptor mutations have been describedfor a subset of these genes, in both mice and humans. In the mouse,disruption of the FGFR1 or FGFR2 genes leads to early embryoniclethality (Deng, et al., Genes Dev. 8:3045-3057, 1994; Yamaguchi, etal., Genes Dev. 8:3032-3044, 1994), and disruption of FGFR3 leads tobone overgrowth (Deng, et al., Cell 84:911-921, 1996; Colvin, et al.,Nature Genet. 12:390-397,1996). In humans, point mutations in FGFR1,FGFR2, and FGFR3 have been found in a variety of skeletal disorders(reviewed by Muenke and Schell, Trends Genet. 11, 308-313, 1995). Recentwork has shown that receptor diversity is increased by alternativepre-mRNA splicing within the extracellular ligand binding domain, withthe result that multiple receptor isoforms, with different ligandbinding properties, can be encoded by the same gene (Johnson andWilliams, supra). In tissue culture systems, binding of AFGF or bFGF toits cell surface receptor activates phospholipase C-gamma (Burgess, etal., Mol. Cell Biol., 10:4770, 1990), which is a component of a pathwayknown to integrate a variety of mitogenic signals. Many members of theFGF family also bind tightly to heparin, and a ternary complex ofheparin, FGF, and a transmembrane receptor may be a biologicallyrelevant signaling species.

SUMMARY OF THE INVENTION

The invention provides fibroblast growth factor homologous factor (FHF)polypeptides and nucleic acids that encode them. FHFs are involved inregulating the growth, survival, and differentiation of cells in thecentral nervous system (CNS), as well as cells in peripheral nervoustissues.

The invention also provides methods for detecting alterations in FHFgene expression, which can be used in the diagnosis of neurodegenerativeand neoplastic disorders. Methods for treating neurodegenerative andneoplastic disorders, in which the expression and/or activity of an FHFis modulated, are also included in the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment o the amino acid sequences of FHF-1 (SEQ IDNO:1), FHF-2 (SEQ ID NO:2), FHF-3 (SEQ ID NO:3), and FHF-4 (SEQ IDNO:4). Amino acids that are conserved in all four of FHFs 1-4 are shadedand boxed and amino acids that are conserved in only some of FHFs 1-4are shaded.

FIG. 2 shows an alignment of the amino acid sequences of FHF-1 (SEQ IDNO:1), FHF-2 (SEQ ID NO:2), FHF-3 (SEQ ID NO:3), FHF-4 (SEQ ID NO:4),FGF-1 (SEQ ID NO:5), FGF-2 (SEQ ID NO:6), and FGF-9 (SEQ ID NO:7). Thelarge, black dots indicate amino acids that are identical among FHFs1-4, but that are different in the nine previously characterized FGFs(FGFs 1-9). Intron locations for murine FHF-2 are indicated byarrowheads above the aligned sequences. The locations of the twelvesegments having beta-sheet conformations in the FGF-2 crystal structureare underlined (Erickson, et al., Proc. Natl. Acad. Sci. USA88:3441-3445, 1991).

FIG. 3 shows an alignment of the amino acid sequences of mouse and humanFHFs with each of the nine, previously characterized members of the FGFfamily (hFHF-1 (SEQ ID NO:1), hFHF-2 (SEQ ID NO:2), hFHF-3 (SEQ IDNO:3), hFHF-4 (SEQ ID NO:4), mFHF-1 (SEQ ID NO:8), mFHF-2 (SEQ ID NO:9),mFHF-3 (SEQ ID NO:10), and mFHF-4 (SEQ ID NO:11)).

The FGF family members include aFGF/FGF-1 (SEQ ID NO:5; Jaye, et al.,supra), bFGF/FGF-2 (SEQ ID NO:6; Abraham, et al., supra), int-2/FGF-3(SEQ ID NO:12; Smith, et al., supra), FGF-4 (SEQ ID NO:13; Delli-Bovi,et al., supra), FGF-5 (SEQ ID NO:14; Zhan, et al., supra), FGF-6 (SEQ IDNO:15; Maricas, et al., supra), keratinocyte growth factor/FGF-7 (SEQ IDNO:16; Finch, et al., supra), FGF-8 (SEQ ID NO:17; Tanaka, et al.,supra), and FGF-9 (SEQ ID NO:7; Miyamoto, et al., supra). (“m” denotesmouse, and “h” denotes human.)

FIG. 4 shows a dendrogram of mammalian FHF and FGF family members, inwhich the length of each path connecting any pair of FHF or FGF familymembers is proportional to the degree of amino acid sequence divergenceof that pair. (“m” denotes mouse, and “h” denotes human.)

FIG. 5 shows the nucleotide (SEQ ID NO:18) and deduced amino acid (SEQID NO:1) sequences of FHF-1.

FIG. 6 shows the nucleotide (SEQ ID NO:19) and deduced amino acid (SEQID NO:2) sequences of FHF-2.

FIG. 7 shows the nucleotide (SEQ ID NO:20) and deduced amino acid (SEQID NO:3) sequences of FHF-3.

FIG. 8 shows the nucleotide (SEQ ID NO:21) and deduced amino acid (SEQID NO:4) sequences of FHF-4.

FIG. 9 shows partial chromosome linkage maps for mouse FHF-1, FHF-2, andFHF-3 genes. The genes were mapped by interspecific backcross analysis.To the left of each chromosome map, the number of recombinant N2 animalsis presented, divided by the total number of N2 animals typed for eachpair of loci. The recombination frequencies, expressed as geneticdistance in centimorgans (+/− one standard error) are also shown. Theupper 95% confidence limit of the recombination distance is given inparentheses, in cases where no recombinants were found between loci. Thepositions of loci on human chromosomes, where known, are shown to theright of the chromosome maps. References for the map positions of mosthuman loci can be obtained from the GDB (Genome Data Base), which is acomputerized database of human linkage information maintained by TheWilliam H. Welch Medical Library of The Johns Hopkins University(Baltimore, Md.).

FIG. 10 shows the tissue distribution of FHF transcripts in the adultmouse. Ten micrograms of total RNA from various mouse tissues wasprepared (Chomczinski and Sacchi, Anal. Biochem., 162:156, 1987) andused in RNAse protection experiments (Ausubel, et al, Current Protocolsin Molecular Biology, Wiley Interscience, New York, N.Y., 1987)employing the indicated antisense riboprobes (FHFs 1-4). The RNA samplesare as follows: 1, brain; 2, eye; 3, heart; 4, kidney; 5, liver; 6,lung; 7, spleen; 8, testis; and 9, yeast tRNA. A control reaction, inwhich an RNA Polymerase II probe was used, is shown at the bottom of thefigure.

FIG. 11 shows in situ localization of FHF transcripts in sectionsprepared from the developing and adult mouse. ³³P in situ hybridizationis shown in red and is superimposed on a cresyl violet stain shown inblack and white. The probes and samples used are as follows: (A) FHF-2,e11; (B) FHF-3, e11; (C, D) FHF-2, e17; (E) FHF-1, P1, coronal sectionthrough the head at the level of the eyes; (F) FHF-2, P1, coronalsection through the center of the head; (G) FHF-1, adult; (H, I) FHF-2,adult; (J) FHF-3, adult; (K, L) FHF-4, adult.

FIG. 12 shows FHF-1 immunostaining in sections of the macaque monkeyCNS. The samples stained are as follows: (A) precentral motor cortex;(B) area 3 b in the primary somatosensory cortex; (C) primary auditorycortex; (D) area 7 b in the superior parietal lobule; and (E) primaryvisual cortex. Panels F-H show that populations of cortical neuronsimmunoreactive for FHF-1 include large intensely, immunoreactive cells(arrows), small, weakly immunoreactive cells (arrowheads), and small,weakly immunoreactive cells, with fine processes resembling microglia(double arrows). Simultaneous immunostaining for parvalbumin (G) andFHF-1 (H) shows that large and small neurons are immunoreactive forboth. An uneven distribution of FHF-1 immunoreactive somata is seen inthe hippocampal formation (I), including the subicular complex (S), theCA fields, and the dentate gyrus (DG). In the dorsal thalamus (J), FHF-1immunoreactive somata occupy patches in the caudal ventroposteriolateralnucleus (VPLc) and are found in both magnocellular and parvicellularlayers of the lateral geniculate nucleus (LGN). The medial geniculatecomplex contains few immunostained cells. In the basal telencephalon(K), immunoreactive neurons are present in both the external andinternal segments of the globus pallidus (Gpe and GPi). Scale bars: 500μm in (A-E), 20 μm in (F), 75 μm in (G, H), and 1 mm in (I-K).

FIG. 13 shows that FHF-1 is not secreted by 293 cells, which are humanembryonic kidney cells. 293 cells were transiently transfected withplasmids directing expression of human growth hormone (left 2 lanes;hGH), FHF-1 (center 2 lanes), or the human red cone pigment (right 2lanes; Red). The cells were labeled for 6 hours with ³⁵S-methionine inserum-free medium, and the total protein present in the cells (C) ormedium (M) was resolved by SDS-PAGE and visualized by autoradiography.Secretion of hGH, but not FHF-1, is observed. The mobilities of proteinstandards are indicated at the left of the figure; from top to bottom,their molecular masses, in kDa, are: 220, 97, 66, 46, 30, 21.5, and14.3. The mobilities of hGH and FHF-1 are indicated at the right side ofthe figure.

FIG. 14 shows a summary of FHF-1 constructs used to identify the FHF-1nuclear localization signal (NLS). Localization of these constructs byimmunostaining (constructs 1-4) and localization ofFHF-1-β-galactosidase fusions by X-gal staining (constructs 5-12) isalso shown. The numbers underneath each construct indicate the aminoacids from FHF-1 that are present in the construct. (N, nuclearstaining; C, cytoplasmic staining; ++, strong staining; +, weakstaining; −, no staining. ‘a’, construct 12 shows cytoplasmic stainingin 15%-20% of cells and nuclear localization in 80%-85% of cells.)

FIG. 15 shows double label immunofluorescent localization of theconstructs illustrated in FIG. 14. The antibodies used are as follows:(A, B) double label immunofluorescent localization of FHF-1 (green) andBiP (an ER marker; red), optically sectioned at 0.7 μm; and (C-F)histochemical localization of FHF-1-β-galactosidase fusion proteins. Allexperiments were performed in transiently transfected 293 cells. Theconstructs used are: (A) full length FHF-1, construct 1; (B) construct4; (C) full length β-galactosidase; (D) construct 6; (E) construct 11;(F) construct 12.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides polypeptide growth factors, designated fibroblastgrowth factor homologous factors (FHFs), and nucleic acids that encodethem. Genes encoding four FHFs, designated FHFs 1-4, have been isolatedand sequenced. The deduced amino acid sequence of FHF-1 is 27% identicalto that of FGF-9, and the amino acid sequences of FHFs 1-4 are 58-70%identical to each other. Thus, FHFs define a new branch of the FGFfamily.

FHFs are expressed in the developing and adult nervous systems, and thusare believed to play roles in regulating nervous system development andfunction. Accordingly, FHF polypeptides, and nucleic acids that encodethem, can be used in methods for treating and diagnosing conditionsaffecting the nervous system, including, e.g., stroke, neurodegenerativediseases, such as Parkinson's disease and Alzheimer's disease, retinaldegenerative diseases, such as retinitis pigmentosa and maculardegeneration, cerebellar degenerative diseases, and cancer. Morespecific uses for FHF-related molecules can be gleaned from their tissuespecificities. For example, although FHFs 1-4 are all expressed in thebrain, FHFs 1-3 are specifically expressed in the eye, FHFs 1 and 4 areexpressed in the testes, and FHF-2 is expressed in the heart. Thus,monoclonal and polyclonal antibodies can be produced using standardimmunization and screening methods well known in the art. Theseantibodies can be easily detectably labelled and used histologically toidentify tissues which contain a given FHF. FHFs can also be used inmethods for maintaining cultured cells or tissues, such as neuronalcells or tissues, prior to transplantation. In addition, FHFs can beused to promote neuron growth in vitro, in order to, for example,facilitate production of growth factors, such as interleukin-2 (IL-2),that are produced by them. Methods employing FHF polypeptides andnucleic acids are described in further detail below.

The invention provides substantially pure FHF polypeptides. FHFpolypeptides can be characterized as containing, for example, at leastfive consecutive amino acids that are conserved in at least two, e.g.,three or four, FHFs, such as FHFs 1-4. One or more (e.g., two to four)of the five conserved amino acids, in addition to being conserved inFHFs, can be characterized as not being conserved in any of the ninepreviously characterized FGFs (FGFs 1-9, see above).

The term “substantially pure” is used herein to describe a molecule,such as a polypeptide (e.g., an FHF polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. One skilled in the art can purify FHF polypeptides usingstandard protein purification methods and the purity of the polypeptidescan be determined using standard methods including, e.g., polyacrylamidegel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., highperformance liquid chromatography (HPLC)), and amino-terminal amino acidsequence analysis.

FHF polypeptides included in the invention can have one of the aminoacid sequences of FHFs 1-4, for example, the amino acid sequence ofFHF-4. FHF polypeptides, such as FHFs 1-4, can be characterized by beingexpressed in the brain, lacking classical signal sequences, containingnuclear localization signals or nuclear localization-like signals, andcontaining, at full length, about 225-250 amino acids (FHF-1:244 aminoacids; FHF-2:245 amino acids; FHF-3:225 amino acids; FHF-4:247 aminoacids; see, e.g., FIGS. 1-3 and 5-8). The FHF polypeptides of theinvention can be derived from a mammal, such as a human or a mouse.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of an FHF polypeptide,such as one of FHFs 1-4, e.g., FHF-4. A “substantially identical” aminoacid sequence is a sequence that differs from a reference sequence onlyby conservative amino acid substitutions, for example, substitutions ofone amino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine), or by one or morenon-conservative substitutions, deletions, or insertions, provided thatthe polypeptide retains at least one FHF-specific activity or anFHF-specific epitope. For example, one or more amino acids can bedeleted from an FHF polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for FHF biological activity, can be removed.Such modifications can result in the development of smaller active FHFpolypeptides.

Other FHF polypeptides included in the invention are polypeptides havingamino acid sequences that are at least 50% identical to the amino acidsequence of an FHF polypeptide, such as any of FHFs 1-4, e.g., FHF-4.The length of comparison in determining amino acid sequence homology canbe, for example, at least 15 amino acids, for example, at least 20, 25,or 35 amino acids. Homology can be measured using standard sequenceanalysis software (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705; also see Ausubel, et al.,supra).

The invention also includes fragments of FHF polypeptides, such as FHFs1-4, that retain at least one FHF-specific activity or epitope. Forexample, an FHF polypeptide fragment containing, e.g., at least 8-10amino acids can be used as an immunogen in the production ofFHF-specific antibodies. The fragment can contain, for example, an aminoacid sequence that is conserved in FHFs, and this amino acid sequencecan contain amino acids that are conserved in FHFs, but not in FGFs 1-9.Such fragments can easily be identified by comparing the sequences ofFHFs and FGFs, e.g., by reference to FIGS. 1-3. In addition to their useas peptide immunogens, the above-described FHF fragments can be used inimmunoassays, such as ELISAs, to detect the presence of FHF-specificantibodies in samples.

The FHF polypeptides of the invention can be obtained using any ofseveral standard methods. For example, FHF polypeptides can be producedin a standard recombinant expression systems (see below), chemicallysynthesized (this approach may be limited to small FHF peptidefragments), or purified from tissues in which they are naturallyexpressed (see, e.g., Ausubel, et al., supra).

The invention also provides isolated nucleic acid molecules that encodethe FHF polypeptides described above, as well as fragments thereof Forexample, nucleic acids that encode any of FHFs 1-4, such as FHF-4, areincluded in the invention. These nucleic acids can contain naturallyoccurring nucleotide sequences (see FIGS. 1-3 and 5-8), or sequencesthat differ from those of the naturally occurring nucleic acids thatencode FHFs 1-4, but encode the same amino acids, due to the degeneracyof the genetic code. The nucleic acids of the invention can contain DNAor RNA nucleotides, or combinations or modifications thereof.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

The nucleic acid molecules of the invention can be used as templates instandard methods for production of FHF gene products (e.g., FHF RNAs andFHF polypeptides; see below). In addition, the nucleic acid moleculesthat encode FHF polypeptides (and fragments thereof) and related nucleicacids, such as (1) nucleic acids containing sequences that arecomplementary to, or that hybridize to, nucleic acids encoding FHFpolypeptides, or fragments thereof (e.g., fragments containing at least12, 15, 20, or 25 nucleotides); and (2) nucleic acids containingsequences that hybridize to sequences that are complementary to nucleicacids encoding FHF polypeptides, or fragments thereof (e.g., fragmentscontaining at least 12, 15, 20, or 25 nucleotides); can be used inmethods focused on their hybridization properties. For example, as isdescribed in further detail below, such nucleic acid molecules can beused in the following methods: PCR methods for synthesizing FHF nucleicacids, methods for detecting the presence of an FHF nucleic acid in asample, screening methods for identifing nucleic acids encoding new FHFfamily members, and therapeutic methods.

The invention also includes methods for identifying nucleic acidmolecules that encode members of the FHF polypeptide family in additionto FHFs 1-4. In these methods, a sample, e.g., a nucleic acid library,such as a cDNA library, that contains a nucleic acid encoding an FHFpolypeptide is screened with an FHF-specific probe, e.g., anFHF-specific nucleic acid probe. FHF-specific nucleic acid probes arenucleic acid molecules (e.g., molecules containing DNA or RNAnucleotides, or combinations or modifications thereof) that specificallyhybridize to nucleic acids encoding FHF polypeptides, or tocomplementary sequences thereof. Because FHFs are closely related toFGFs (i.e., the first nine members of the FGF family (FGFs 1-9), seeabove), the term “FHF-specific probe,” in the context of this method ofinvention, refers to probes that bind to nucleic acids encoding FHFpolypeptides, or to complementary sequences thereof, to a detectablygreater extent than to nucleic acids encoding FGFs, or to complementarysequences thereof. The term “FHF-specific probe” thus includes probesthat can bind to nucleic acids encoding FHF polypeptides (or tocomplementary sequences thereof), but not to nucleic acids encoding FGFs(or to complementary sequences thereof), to an appreciable extent.

The invention facilitates production of FHF-specific nucleic acidprobes. Methods for obtaining such probes can be designed based on theamino acid sequence alignments shown in FIGS. 1-3. In FIG. 1, forexample, amino acid sequences that are conserved in FHFs (“FHF-conservedamino acids”) are boxed. In FIG. 2, amino acids that are conserved inFHFs, but not in FGFs (i.e., FGFs 1-9) (“FHF-specific amino acids”), areindicated by large, black dots. The probes, which can contain at least12, e.g.,at least 15, 25, 35, 50, 100, or 150 nucleotides, can beproduced using any of several standard methods (see, e.g., Ausubel, etal., supra). For example, preferably, the probes are generated using PCRamplification methods, such as those described below in Example 1. Inthese methods, primers are designed that correspond to FHF-conservedsequences (FIG. 1), which can include FHF-specific amino acids, and theresulting PCR product is used as a probe to screen a nucleic acidlibrary, such as a cDNA library. A nucleotide sequence encoding FHF-4was identified generally following this process based upon the analysisof the sequences of FHF 1-3.

As is known in the art, PCR primers are typically designed to contain atleast 15 nucleotides, for example 15-30 nucleotides. The design ofFHF-specific primers containing 21 nucleotides, which encode FHFpeptides containing 7 amino acids, are described as follows. Preferably,most or all of the nucleotides in such a probe encode FHF-conservedamino acids, including FHF-specific amino acids. For example, primerscontaining sequences encoding peptides containing at least 40%FHF-conserved amino acids can be used. Such a primer, containing 21nucleotides, can include sequences encoding at least 3/7, 4/7, 5/7, 6/7,or 7/7 FHF-conserved amino acids. As can be determined by analysis ofFIGS. 1-3, in the case of a 21 nucleotide primer, encoding 7 aminoacids, up to 5 amino acids can be FHF-specific. Thus, the primer cancontain sequences encoding at least one FHF-specific amino acid, forexample, up to 5 FHF-specific amino acids. Once FHF-specific amino acidsequences are selected as templates against which primer sequences areto be designed, the primers can be synthesized using, e.g., standardchemical methods. As is described above, due to the degeneracy of thegenetic code, such primers should be designed to include appropriatedegenerate sequences, as can readily be determined by one skilled in theart (see above, and Example 1, below).

Based on the guidelines presented above, examples of FHF-conserved aminoacid peptides that can be used as templates for the design ofFHF-specific primers are as follows. Additional examples can be found byanalysis of sequence alignments of FHF polypeptides, for example, thealignments in FIGS. 1-3. Primers can be designed, for example, based on5-10 amino acid regions of these peptides, depending on the lengths ofthe primers desired. For example, primers can be designed to correspondto 7 consecutive amino acids of any of the segments shown below.

1. AAAI/LASS/GSLIRQKR (SEQ ID NO:22) (corresponding to amino acids 2-14of human FHF-1)

2. PQLKGIVTR/K (SEQ ID NO:23) (corresponding to amino acids 68-76 ofhuman FHF-1)

3. TL/HFNLIPVGLRVV (SEQ ID NO:24) (corresponding to amino acids 104-116of human FHF-1)

4. AMNG/S/AEGY/LLY (SEQ ID NO:25) (corresponding to amino acids 128-136of human FHF-1)

5. KES/CVFENYYV (SEQ ID NO:26) (corresponding to amino acids 148-157 ofhuman FHF-1; see Example 1, below, for an example of a primer based onthe “VFENYYV” (SEQ ID NO:27) portion of this sequence.)

6. SGRA/GWF/YLGL (SEQ ID NO:28) (corresponding to amino acids 169-177 ofhuman FHF-1)

7. MKGNR/HVKKT/NK (SEQ ID NO:29) (corresponding to amino acids 184-193of human FHF-1; see Example 1, below, for an example of a primer basedon the “MKGNH/RVK” (SEQ ID NO:30) portion of this sequence.)

8. VC/AMYR/Q/KEPSLH (SEQ ID NO:31) (corresponding to amino acids 205-214of human FHF-1)

As is described above, FHF-specific r\primers, for example primers basedon the FHF-specific peptides shown above, or portions thereof, can beused in PCR reactions to generate FHF-specific probes, which can be usedin standard screening methods to identify nucleic acids encoding FHFfamily members (see, e.g., Ausubel, et al., supra).

In addition to FHF-specific nucleic acid probes, FHF-specificpolypeptide probes, such as FHF-specific antibodies, can be used toscreen samples, e.g., expression libraries, for nucleic acids encodingnovel FHF polypeptides, or portions thereof. For example, an antibodythat specifically binds to an FHF-specific peptide can be used in thismethod. Methods for carrying out such screening are well known in theart (see, e.g. Ausubel, et al., supra).

The sequences of a pair of nucleic acid molecules (or two regions withina single nucleic acid molecule) are said to be “complementary” to eachother if base pairing interactions can occur between each nucleotide ofone of the members of the pair and each nucleotide of the other memberof the pair. A pair of nucleic acid molecules (or two regions within asingle nucleic acid molecule) are said to “hybridize” to each other ifthey form a duplex by base pairing interactions between them. As isknown in the art, hybridization between nucleic acid pairs does notrequire complete complementarity between the hybridizing regions, butonly that there is a sufficient level of base pairing to maintain theduplex under the hybridization conditions used.

Hybridization reactions are typically carried out under low to moderatestringency conditions, in which specific and some non-specificinteractions can occur. After hybridization, washing can be carried outunder moderate or high stringency conditions to eliminate non-specificbinding. As is known in the art, optimal washing conditions can bedetermined empirically, e.g., by gradually increasing the stringency.Condition parameters that can be changed to affect stringency include,e.g., temperature and salt concentration. In general, the lower the saltconcentration and the higher the temperature, the higher the stringency.For example, washing can be initiated at a low temperature (e.g., roomtemperature) using a solution containing an equivalent or lower saltconcentration as the hybridization solution. Subsequent washing can becarried out using progressively warmer solutions having the same saltsolution. Alternatively, the salt concentration can be lowered and thetemperature maintained in the washing step, or the salt concentrationcan be lowered and the temperature increased. Additional parameters canbe altered to affect stringency, including, e.g., the use of adestabilizing agent, such as formamide.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter. An exampleof progressively higher stringency conditions is as follows: 2×SSC/0.1%SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1%SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1%SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC atabout 68° C. (high stringency conditions). Washing can be carried outusing only one of these conditions, e.g., high stringency conditions, oreach of the conditions can be used, e.g., for 10-15 minutes each, in theorder listed above, repeating any or all of the steps listed. However,as mentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

The nucleic acid molecules of the invention can be obtained by any ofseveral standard methods. For example, the molecules can be producedusing standard recombinant, enzymatic (e.g., PCR or reversetranscription (RT)/PCR methods), and chemical (e.g.,phosphoramidite-based synthesis) methods. In addition, they can beisolated from samples, such as nucleic acid libraries and tissuesamples, using standard hybridization methods. For example, as describedabove, using standard methods, genomic or cDNA libraries can behybridized with nucleic acid probes corresponding to FHF nucleic acidsequences to detect the presence of a homologous nucleotide sequence inthe library (see, e.g., Ausubel, et al., supra). These methods aredescribed in more detail above. Also as described above, nucleic acidsencoding polypeptides containing at least one FHF epitope, such as anFHF-specific epitope, can also be identified by screening a cDNAexpression library, such as a library contained in lambda gt11, with anFHF-specific antibody as a probe. Such antibodies can be eitherpolyclonal or monoclonal and are produced using standard methods (see,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

The FHF nucleic acid molecules can be inserted into vectors, such asplasmid or viral vectors, that facilitate (1) expression of the insertednucleic acid molecule and/or (2) amplification of the insert. As is wellknown in the art, such vectors can contain, e.g., promoter sequences,which facilitate transcription of the inserted nucleic acid in the cell,origins of replication, and genes, such as a neomycin-resistance gene,which encodes a selectable marker that imparts G418 resistance to cellsin which it is expressed, and thus permits phenotypic selection oftransformed cells.

Vectors suitable for use in the present invention include, e.g.,T7-based expression vectors for use in bacteria (see, e.g., Rosenberg,et al., Gene, 56:125, 1987), the pMSXND expression vector for use inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988), andbaculovirus-derived vectors for use in insect cells. The nucleic acidsin such vectors are operably linked to a promoter, which is selectedbased on, e.g., the cell type in which expression is sought. Forexample, a T7 promoter can be used in bacteria, a polyhedrin promotercan be used in insect cells, and a cytomegalovirus or metallothioneinpromoter can be used in mammalian cells. Also, in the case of highereukaryotes, tissue-specific promoters are available. (See, e.g.,Ausubel, et al., supra, for additional appropriate vectors and promotersthat can be used in the invention; also see Pouwels, et al.; CloningVectors: A Laboratory Manual, 1985, Supp. 1987). Viral vectors that canbe used in the invention include, for example, retroviral, adenoviral,adeno-associated viral, herpes virus, simian virus 40 (SV40), and bovinepapilloma virus vectors (see, e.g., Gluzman ed., Eukaryotic ViralVectors, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1982), and are discussed further below.

Cells into which FHF nucleic acids can be introduced, in order to, forexample, produce FHF polypeptides using, e.g., the vectors describedabove, include prokaryotic cells (e.g., bacterial cells, such as E. colicells) and eukaryotic cells (e.g., yeast cells, such as Saccharomycescerevisiae cells; insect cells, such as Spodoptera frugiperda cells(e.g., Sf-9 cells); and mammalian cells, such as CHO, Cos-1, NIH-3T3,and JEG3 cells). Such cells are available from a number of differentsources that are known to those skilled in the art, e.g., the AmericanType Culture Collection (ATCC), Rockville, Md. (also see Ausubel, etal., supra). Cells into which the nucleic acids of the invention havebeen introduced, as well as their progeny, even if not identical to theparental cells, due to mutations, are included in the invention.

Methods for introducing the nucleic acids of the invention (e.g.,nucleic acids inserted into the vectors described above) into cells,either transiently or stably, are well known in the art (see, e.g.,Ausubel, et al., supra). For example, in the case of prokaryotic cells,such as E. coil cells, competent cells, which are prepared fromexponentially growing bacteria using a standard CaCl₂ (or MgCl₂ or RbCl)method, can be transformed using standard methods. Transformation ofbacterial cells can also be performed using protoplast fusion methods.In the case of eukaryotic cells, transfection can be carried out usingcalcium phosphate precipitation or conventional mechanical procedures,such as microinjection and electroporation, can be used. Also, thenucleic acid (e.g., contained in a plasmid) can be packaged in aliposome using standard methods. In the case of viral vectors,appropriate infection methods, which are well known in the art, can beused (see, e.g., Ausubel, et al., supra). In addition to beingtransfected with a nucleic acid encoding an FHF polypeptide of theinvention, eukaryotic cells, such as mammalian cells, can beco-transfected with a second nucleic acid encoding a selectable marker,such as a neomycin resistance gene or the herpes simplex virus thymidinekinase gene. As is mentioned above, such selectable markers canfacilitate selection of transformed cells.

Isolation and purification of polypeptides produced in the systemsdescribed above can be carried out using conventional methods,appropriate for the particular system. For example, preparativechromatography and immunological separations employing antibodies, suchas monoclonal or polyclonal antibodies, can be used.

Antibodies, such as monoclonal and polyclonal antibodies, thatspecifically bind to FHF polypeptides (e.g., any or all of FHFs 1-4) arealso included in the invention. These antibodies can be made by using anFHF polypeptide, or an FHF polypeptide fragment that maintain an FHFepitope, as an immunogen in standard antibody production methods (see,e.g., Kohler, et al., Nature, 256:495, 1975; Ausubel, et al., supra;Harlow and Lane, supra).

The term “antibody,” as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)₂, Fv, and SCA fragments, that are capable of bindingto an epitope of an FHF polypeptide. These antibody fragments, whichretain some ability to selectively bind to the antigen (e.g., an FHFantigen) of the antibody from which they are derived, can be made usingwell known methods in the art (see, e.g., Harlow and Lane, supra), andare described further, as follows.

(1) A Fab fragment consists of a monovalent antigen-binding fragment ofan antibody molecule, and can be produced by digestion of a wholeantibody molecule with the enzyme papain, to yield a fragment consistingof an intact light chain and a portion of a heavy chain.

(2) A Fab′ fragment of an antibody molecule can be obtained by treatinga whole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

(3) A (Fab′)₂ fragment of an antibody can be obtained by treating awhole antibody molecule with the enzyme pepsin, without subsequentreduction. A (Fab′)₂ fragment is a dimer of two Fab′ fragments, heldtogether by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

(5) A single chain antibody (“SCA”) is a genetically engineered singlechain molecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as an FHF polypeptide, to which theparatope of an antibody, such as an FHF-4-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics.

As is mentioned above, antigens that can be used in producingFHF-specific antibodies include FHF polypeptides, e.g., any of FHFs 1-4,or FHF polypeptide fragments. The polypeptide or peptide used toimmunize an animal can be obtained by standard recombinant, chemicalsynthetic, or purification methods. As is well known in the art, inorder to increase immunogenicity, an antigen can be conjugated to acarrier protein. Commonly used carriers include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid. The coupled peptide is then used to immunize the animal (e.g., amouse, a rat, or a rabbit). In addition to such carriers, well knownadjuvants can be administered with the antigen to facilitate inductionof a strong immune response.

FHF-specific polyclonal and monoclonal antibodies can be purified, forexample, by binding to, and elution from, a matrix containing an FHFpolypeptide, e.g., the FHF polypeptide (or fragment thereof) to whichthe antibodies were raised. Additional methods for antibody purificationand concentration are well known in the art and can be practiced withthe FHF-specific antibodies of the invention (see, for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994).

Anti-idiotype antibodies corresponding to FHF-specific antigens are alsoincluded in the invention, and can be produced using standard methods.These antibodies are raised to FHF-specific antibodies, and thus mimicFHF-specific epitopes to.

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

As is discussed above, because of their amino acid sequence homologiesto previously identified FGF polypeptides (i.e., FGFs 1-9), as well astheir tissue localizations, FHFs are thought to play roles in regulatingthe development and function of the nervous system. Altered levels ofFHFs, such as increased levels, may thus be associated with cellproliferative disorders, such as cell proliferative disorders of thenervous system. The term “cell-proliferative disorder” is used herein todescribe conditions that are characterized by abnormally excessive cellgrowth, including malignant, as well as non-malignant, cell growth.Conversely, conditions characterized by inadequate cell growth may becharacterized by decreased expression of FHFs. Accordingly, theseconditions can be diagnosed and monitored by detecting the levels ofFHFs in patient samples.

FHF-specific antibodies and nucleic acids can be used as probes inmethods to detect the presence of an FHF polypeptide (using an antibody)or nucleic acid (using a nucleic acid probe) in a sample, such as abiological fluid (e.g., cerebrospinal fluid (CSF), such as lumbar orventricular CSF) or a tissue sample (e.g., CNS tissue, e.g., neuraltissue or eye tissue). In these methods, an FHF-specific antibody ornucleic acid probe is contacted with a sample from a patient suspectedof having an FHF-associated disorder, and specific binding of theantibody or nucleic acid probe to the sample detected. The level of FHFpolypeptide or nucleic acid present in the suspect sample can becompared with the level in a control sample, e.g., an equivalent samplefrom an unaffected individual, to determine whether the patient has anFHF-associated cell proliferative disorder. FHF polypeptides, orfragments thereof, can also be used as probes in diagnostic methods, forexample, to detect the presence of FHF-specific antibodies in samples.

The FHF-specific nucleic acid probes can be labeled with a compound thatfacilitates detection of binding to the FHF nucleic acid in the sample.For example, the probe can contain biotinylated nucleotides, to whichdetectably labeled avidin conjugates (e.g., horse-radishperoxidase-conjugated avidin) can bind. Radiolabeled nucleic acid probescan also be used. These probes can be used in nucleic acid hybridizationassays to detect altered levels of FHFs in a sample. For example, insitu hybridization, RNAse protection, and Northern Blot methods can beused. Other standard nucleic acid detection methods that can be used inthe invention are known to those of skill in the art (see, e.g.,Ausubel, et al., supra). In addition, when the diagnostic molecule is anucleic acid, it can be amplified prior to binding with an FHF-specificprobe. Preferably, PCR is used, but other nucleic acid amplificationmethods, such as the ligase chain reaction (LCR), ligated activatedtranscription (LAT), and nucleic acid sequence-based amplification(NASBA) methods can be used.

Use of FHF-specific antibodies in diagnostic methods is describedfurther, as follows. The antibodies of the invention can be used invitro or in vivo for immunodiagnosis. The antibodies are suited for usein, for example, immunoassays in which they are in liquid phase or boundto a solid phase carrier (e.g., a glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylase, natural and modified cellulose,polyacrylamide, agarose, or magnetite carrier). The antibodies used insuch immunoassays can be detectably labeled (e.g., with an enzyme, aradioisotope, a fluorescent compound, a colloidal metal, achemi-luminescent compound, a phosphorescent compound, or abioluminescent compound) using any of several standard methods that arewell known in the art. Examples of immunoassays in which the antibodiesof the invention can be used include, e.g., competitive andnon-competitive immunoassays, which are carried out using either director indirect formats. Examples of such immunoassays includeradioimmunoassays (RIA) and sandwich assays (e.g., enzyme-linkedimmunosorbent assays (ELISAs)). Detection of antigens using theantibodies of the invention can be done using immunoassays that are runin either forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Other immunoassayformats are well known in the art, and can be used in the invention(see, e.g., Coligan, et al., supra).

In addition to the in vitro methods described above, FHF-specificmonoclonal antibodies can be used in methods for in vivo detection of anantigen, such as an FHF antigen (e.g., any one of FHFs 1-4). In thesemethods, a detectably labeled antibody is administered to a patient in adose that is determined to be diagnostically effective by one skilled inthe art. The term “diagnostically effective” is used herein to describethe amount of detectably labeled monoclonal antibody that isadministered in a sufficient quantity to enable detection of the sitehaving the antigen for which the monoclonal antibody is specific. Aswould be apparent to one skilled in the art, the concentration ofdetectably labeled monoclonal antibody that is administered should besufficient so that the binding of the antibody to the cells containingthe polypeptide is detectable, compared to background. Further, it isdesirable that the detectably labeled monoclonal antibody is rapidlycleared from the circulatory system, to give the optimaltarget-to-background signal ratio.

The dosage of detectably labeled monoclonal antibodies for in vivodiagnosis will vary, depending on such factors as the age and weight ofthe individual, as well as the extent of the disease. The dosages canalso vary depending on factors such as whether multiple administrationsare intended, antigenic burden, and other factors known to those ofskill in the art.

In addition to initial diagnosis, the FHF polypeptides, nucleic acids,and FHF-specific antibodies described above can be used in in vitro orin vivo methods for monitoring the progress of a condition associatedwith FHF expression. For example, they can be used in methods to monitorthe course of amelioration of an FHF-associated disease, for example,after treatment has begun. In these methods, changes in the levels of anFHF-specific marker (e.g., an FHF polypeptide, an FHF nucleic acid, oran FHF-specific antibody) are detected, either in a sample from apatient or using the in vivo methods described above.

The invention also provides methods for treating conditions associatedwith altered expression of FHF polypeptides, for example, cellproliferative disorders (e.g., cell proliferative disorders of thecentral or peripheral nervous systems, for example, conditions affectingneural tissue, testes, heart tissue, and cells of the eye). Treatment ofan FHF-associated cell proliferative disorder can be carried out, forexample, by modulating FHF gene expression or FHF activity in a cell.The term “modulate” includes, for example, suppressing expression of anFHF when it is over-expressed, and augmenting expression of an FHF whenit is under-expressed. In cases where a cell-proliferative disorder isassociated with over-expression of an FHF, nucleic acids that interferewith FHF expression, at transcriptional or translational levels, can beused to treat the disorder. This approach employs, for example,antisense nucleic acids (i.e., nucleic acids that are complementary to,or capable of hybridizing with, a target nucleic acid, e.g., a nucleicacid encoding an FHF polypeptide), ribozymes, or triplex agents. Theantisense and triplex approaches function by masking the nucleic acid,while the ribozyme strategy functions by cleaving the nucleic acid. Inaddition, antibodies that bind to FHF polypeptides can be used inmethods to block the activity of an FHF.

The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (see, e.g., Marcus-Sakura, Anal.Biochem., 12:289, 1988). Antisense nucleic acids are nucleic acidmolecules (e.g., molecules containing DNA nucleotides, RNA nucleotides,or modifications (e.g., modification that increase the stability of themolecule, such as 2′—O-alkyl (e.g., methyl) substituted nucleotides) orcombinations thereof) that are complementary to, or that hybridize to,at least a portion of a specific nucleic acid molecule, such as an RNAmolecule (e.g., an mRNA molecule) (see, e.g., Weintraub, ScientificAmerican, 262:40, 1990). The antisense nucleic acids hybridize tocorresponding nucleic acids, such as mRNAs, to form a double-strandedmolecule, which interferes with translation of the mRNA, as the cellwill not translate an double-stranded mRNA. Antisense nucleic acids usedin the invention are typically at least 10-12 nucleotides in length, forexample, at least 15, 20, 25, 50, 75, or 100 nucleotides in length. Theantisense nucleic acid can also be as long as the target nucleic acidwith which it is intended that it form an inhibitory duplex. As isdescribed further below, the antisense nucleic acids can be introducedinto cells as antisense oligonucleotides, or can be produced in a cellin which a nucleic acid encoding the antisense nucleic acid has beenintroduced by, for example, using gene therapy methods.

Introduction of FHF antisense nucleic acids into cells affected by aproliferative disorder, for the purpose of gene therapy, can be achievedusing a recombinant expression vector, such as a chimeric virus or acolloidal dispersion system, such as a targeted liposome. Those of skillin this art know or can easily ascertain the appropriate route and meansfor introduction of sense or antisense FHF nucleic acids, without resortto undue experimentation.

Gene therapy methods can also be used to deliver genes encoding FHFpolypeptides (e.g., any of FHFs 1-4) to cells. These methods can becarried out to treat conditions associated with insufficient FHFexpression. Thus, these methods can be used to promote tissue repair orreplacement, for example, in conditions including stroke,neurodegenerative diseases, such as Parkinson's disease and Alzheimer'sdisease, retinal degenerative diseases, such as retinitis pigmentosa andmacular degeneration, and peripheral neuropathies.

Due to the high levels of expression of FHF-4 in the testes, there are avariety of applications for FHF-4-specific polypeptides, nucleic acids,and antibodies related to treating disorders of this tissue. Suchapplications include treatment of cell proliferative disorders relatedto FHF-4 expression in the testes. Various testicular developmental oracquired disorders can also be treated using FHF-4-related molecules.These conditions include, for example, viral infection (e.g., viralorchitis), autoimmunity, sperm production or dysfunction, trauma, andtesticular tumors. The presence of high levels of FHF-4 in the testesalso suggests that FHF-4, or an FHF-4 analogue, can be used to affectmale fertility.

In addition to blocking mRNA translation, oligonucleotides, such asantisense oligonucleotides, can be used in methods to stalltranscription, such as the triplex method. In this method, anoligonucleotide winds around double-helical DNA in a sequence-specificmanner, forming a three-stranded helix, which blocks transcription fromthe targeted gene. These triplex compounds can be designed to recognizea unique site on a chosen gene (Maher, et al., Antisense Res. and Dev.,1(3):227, 1991; Helene, Anticancer Drug Design, 6(6):569, 1991).Specifically targeted ribozymes can also be used in therapeutic methodsdirected at decreasing FHF expression.

The following examples are intended to illustrate, but not to limit, theinvention. While the procedures described in the examples are typical ofthose that can be used to carry out certain aspects of the invention,other procedures known to those skilled in the art can also be used. Thefollowing materials and methods were used in carrying out theexperiments described in the examples.

MATERIALS AND METHODS

Random cDNA Sequencing. Details of retina cDNA library construction,template preparation, and sequence determination are described by Wang,et al., J. Biol. Chem. 271, 4468-4476, 1996.

Degenerate PCR. A fully degenerate sense strand primer, with a flankingEcoRI restriction site, was synthesized to correspond to the amino acidsequence VFENYYV (SEQ ID NO:27), and three partially degenerateantisense primers, with a flanking BamHI site, were synthesized toinclude all possible codons for the amino acid sequence MKGN(H/R)VK (SEQID NO:30). These primers were used to amplify human, murine, and bovinegenomic DNA templates using T. aquaticus polymerase under the followingconditions: 1×(94° C., 7 minutes), 35×(45° C., 2 minutes; 72° C., 0.5minutes; 94° C., 0.5 minutes; 95° C., 0.25 minutes). PCR products werecleaved with EcoRI and BamHI, fractionated by preparative agarose gelelectrophoresis, subcloned into pBluescript, and sequenced individually.

cDNA and Genomic Clones. Oligo-dT primed cDNA libraries from adult humanretinas (Nathans, et al., Science 232, 193-202, 1986) and P0-P7 mouseeyes were screened by DNA hybridization under standard conditions (see,e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The complete coding region sequences of human FHF-1, FHF-2, and FHF-3,and mouse FHF-1, FHF-3, and FHF-4 were obtained from two independentcDNA clones. For human FHF-4, the first 72 codons were sequenced from asingle clone, and the rest of the coding region was sequenced from twoindependent clones. For mouse FHF-2, the coding sequence was determinedfrom cloned genomic DNA and from a PCR product obtained by amplificationof the full-length, coding region from the P0-P7 mouse eye cDNA library.A partial MboI digest of a mouse genomic DNA library in bacteriophagelambda was screened to obtain the mouse FHF-2 genomic clones.

Chromosomal Localization. Human chromosome mapping was performed bySouthern blot analysis of DNA obtained from a panel of 24 human-mouse orhuman-hamster hybrid cell lines each carrying a different humanchromosome (Oncor, Gaithersburg, Md.). Interspecific backcross progenywere generated by mating (C57BL/6J×M. spretus) F1 females and C57BL/6Jmales, as described (Copeland and Jenkins, Trends Genet. 7, 113-118,1991). A total of 205 N2 mice were used to map the FHF loci. DNAisolation, restriction enzyme digestion, agarose gel electrophoresis,Southern blot transfer, and hybridization were performed essentially asdescribed (Jenkins, et al., J. Virol. 43, 26-36, 1982), using Zetabindnylon membranes (AMF-Cuno). Washing was carried out to a finalstringency of 0.8-1.0×SSCP, 0.1% SDS, at 65° C. The FHF-1 probe, a 1kilobase fragment of mouse genomic DNA, detected fragments of 2.1kilobases in C57BL/6J (B) DNA and 10.0 kilobases in M. spretus (S) DNA,following digestion with BamHI. The FHF-2 probe, a 0.75 kilobasefragment of mouse cDNA, detected BglI fragments of 19.0, 11.5, 8.2, and2.2 kilobases (B) and 13.5, 8.2, and 2.2 kilobases (S). The FHF-4 probe,a 0.3 kilobase fragment of mouse cDNA, detected EcoRV fragments ofapproximately 24.0 kilobases (B) and 7.7 kilobases (S).

Most of the probes and RFLPs for the loci linked to the FHF genes in theinterspecific backcross have been reported earlier. These include: Irg1and Rap2a on chromosome 14 (Lee, et al., Immunogenet. 41, 263-270,1995); Smst on chromosome 16 (Siracusa, et al., Genetics 127, 169-179,1991); and Hprt, Cd401, and Ar on the X chromosome (Allen, et al.,Science 259, 990-993, 1993; Fletcher, et al., Genomics 24, 127-132,1994). The probe for apolipoprotein D (Apod), a 290 base pairHindIII/BamHI fragment of a rat cDNA that was provided by Alan Peterson,detected XbaI fragments of 3.1 and 2.8 kilobases (B) and 3.1 and 2.6kilobases (S). The inheritance of the 2.6 kilobase M. spretus-specificXbaI RFLP was followed. Recombination distances were calculated asdescribed (Green, in Genetics and Probability in Animal BreedingExperiments (Oxford Press, New York), pp. 77-113, 1981) using thecomputer program SPRETUS MADNESS. Gene order was determined byminimizing the number of recombination events required to explain theallele distribution patterns.

RNAse Protection. Total RNA was prepared from adult mouse brain, eye,heart, kidney, liver, lung, spleen, and testis by homogenization inguanidinium thiocyanate and extraction with phenol, followed bycentrifugation through 5.7 M cesium chloride (Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Ten micrograms oftotal RNA from each tissue, or ten micrograms of yeast tRNA, was usedfor the RNAse protection assay. Riboprobes were synthesized using eitherT7 or T3 RNA polymerase on linearized templates that were cloned inpBluescript. Each mouse FHF probe contained 150-250 nucleotides from theantisense strand, linked to 25-50 nucleotides of vector sequence.Reagents were obtained from Ambion (Austin, Tex.), and the hybridizationand digestion conditions used were as recommended by Ambion.

In Situ Hybridization. Freshly dissected adult mouse brains, wholeembryos, or heads were rapidly frozen in plastic molds placed on a dryice/ethanol slurry and processed for sectioning as previously described(Cole, et al., J. Neurochem. 55, 1920-1927, 1990). ³³P-labeled antisenseriboprobes were prepared from linearized pBluescript plasmid subclones,using either T3 or T7 RNA polymerase. In situ hybridization wasperformed in 50% formamide, 0.3 M NaCl at 56° C., as described (Saffen,Proc. Natl. Acad. Sci. USA 85, 7795-7799, 1988). Following RNAsetreatment, the slides were washed for 1 hour in 0.1×SSC at 55° C. Afterthe hybridized sections were exposed to X-ray film, the slides werestained with cresyl violet. Digitized images of the stained slides andcorresponding autoradiograms were superimposed using Adobe Photoshopsoftware. The probes used were: FHF-1, 0.75 kilobase containing thecomplete coding region; FHF-2, 0.5 kilobase containing 0.3 kilobase ofintron 4 and 0.2 kilobase of exon 5; FHF-3, two 0.4 kilobase segmentscontaining the 5′ or 3′ halves of the coding region; and FHF-4, 0.5kilobase containing the 3′ two-thirds of the coding region. The codingregions of the different murine FHFs share between 63% and 71%nucleotide sequence identity, suggesting that there should be little orno cross-hybridization under the conditions used.

Immunohistochemistry. Rabbit polyclonal anti-FHF-1 antibodies wereraised against a bacterial fusion protein consisting of thecarboxyl-terminal 190 amino acids of FHF-1 fused to the T7 gene 10protein (Studier, et al., Meth. Enzymol. 185, 60-89, 1980). Anti-FHF-1antibodies were affinity purified using the fusion protein immobilizedon nitrocellulose as an affinity matrix, and antibodies directed againstthe fusion partner were removed by absorption onto immobilized T7 gene10 protein. For immunostaining of primate brain samples, three monkeys(Macaca mulatta) were anesthetized with

Ketamine (35 mg/kg, i.m.), injected with a lethal dose of sodiumpentobarbital (100 mg/kg, i.v.), and perfused through the heart with 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains wereremoved, cryoprotected in 20% sucrose at 4° C., frozen, and cut at athickness of 10 or 20 μm. Sections were sequentially incubated withaffinity purified-rabbit anti-FHF-1 antibodies, biotinylated goatanti-rabbit IgG (Vector), peroxidase-conjugated avidin (Extravidin,Sigma Chemical Co.), and 3,3′-diaminobenzidine dihydrochloride(Aldrich), in the presence of hydrogen peroxide. Most sections were thenmounted on gelatin-coated slides, dehydrated, cleared, and covered witha cover-slip. Following the histochemical reaction, some of the 10μm-thick sections were washed and incubated sequentially in mouseanti-parvalbumin (Sigma), biotinylated horse anti-mouse IgG (Vector),and streptavidin conjugated to Texas Red (Chemicon). These sections weremounted on clean slides, partially dried, and covered in aglycerol/phosphate buffer medium. Adjacent sections were processedhistochemically for cytochrome oxidase or were stained for Nisslsubstance, to determine the boundaries of subcortical nuclei or corticalareas and layers.

Production and Localization of FHF-1 in Transfected Cells. To increasethe efficiency of FHF-1 translation, the region immediately 5′ to theinitiator methionine coding sequence was converted to an optimalribosome binding site (CCACCATGG) by PCR amplification, before insertingthe complete FEH-1 open reading frame into the eukaryotic expressionvector pCIS (Gorman, et al., DNA Protein Eng. Tech. 2, 3-10, 1990). ThepCIS vector was also used for the beta-galactosidase constructs. Humanembryonic kidney cells (293 cells) were transiently transfected with theexpression construct and a plasmid expressing the simian virus 40 (SV40)large T-antigen (pRSV-TAg) using the calcium phosphate method (Gorman,et al., supra). For ³⁵S-methionine labeling, cells were transferred toserum-free medium 24 hours after transfection, and labeled for 6 hours.For immunostaining, transfected cells were grown on gelatin-coatedcoverslips. One day after transfection, the cells were fixed in 2%formaldehyde in PBS for 30 minutes at room temperature, permeabilizedwith cold methanol for 10 minutes at −20° C., preincubated for 15minutes in 3% BSA in phosphate-buffered saline (PBS), incubated for 1hour at room temperature with affinity purified anti-FHF-1 antibody at adilution of 1:1000 and mouse monoclonal anti-BiP at a dilution of 1:400in 3% BSA in PBS, washed 3×15 minutes with 0.1% TWEEN-20™ in PBS at roomtemperature, and then incubated with fluorescein-conjugated donkeyanti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG in 3% BSAin PBS for 30-60 minutes. The coverslips were then washed in 0.1%TWEEN-20™ in PBS and mounted in 0.1% 1,4-diazobicyclo-[2,2,2]-octane(DABCO), 75% glycerol, 10 mM Tris, pH 8.0. For X-gal staining,transfected cells were fixed in 0.5% glutaraldehyde/PBS for 10 minutesat room temperature, washed twice in PBS with 2 mM MgCl₂, incubated in 1mg/ml 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 2 mM MgCl₂ in PBS for 1-2 hours at37° C. and postfixed in 0.5% glutaraldehyde in PBS for 10 minutes atroom temperature.

EXAMPLE 1 IDENTIFICATION OF FIBROBLAST GROWTH FACTOR HOMOLOGOUS FACTORS(FHFS)

To identify gene products expressed in the human retina, random segmentsof human retina cDNA clones were partially sequenced, and the resultingpartial sequences were compared to sequences in publicly availabledatabases. In detail, an adult human retina cDNA library constructed inlambda gt10 (Nathans, et al., Science, 232:193, 1986) was amplified, andthe cDNA inserts were excised en mass by cleavage with EcoRI andpurified from the vector by agarose gel electrophoresis. Following heatdenaturation of the purified cDNA inserts, a synthetic oligonucleotidehaving an EcoRI site at its 5′ end and six random nucleotides at its 3′end (5′-GACGAGATATTAGAATTCTACTCGNNNNNN-3′; (SEQ ID NO:32)) was used toprime two sequential rounds of DNA synthesis in the presence of theKlenow fragment of E. Coli DNA polymerase. The resulting duplex DNAmolecules were amplified by the polymerase chain reaction (PCR) using aprimer corresponding to the unique 5′ flanking sequence(5′-CCCCCCCCCGACGAGATATTAGAATTCTACTCG-3′; (SEQ ID NO:33)). The PCRproducts, representing a random sampling of the original cDNA inserts,were cleaved with EcoRI, size fractionated by preparative agarose gelelectrophoresis to include only segments of approximately 500 base pairsin length, and cloned into lambda gt10. Three thousand single plaquesfrom this library were arrayed in 96-well trays and the inserts fromthese clones were amplified by PCR and then sequenced using the dideoxymethod and automated fluorescent detection (Applied Biosystems). Asingle sequencing run from one end of each insert was conceptuallytranslated for both strands, in all three reading frames, and the sixresulting amino acid sequences were used to search for homology in theGenBank nonredundant protein database using the BLASTX searchingalgorithm.

One partial cDNA sequence was identified that showed statisticallysignificant homology to previously described members of the FGF family.Using this partial cDNA as a probe, multiple, independent cDNA cloneswere isolated from the human retina cDNA library, including two clonesthat encompass the entire open reading frame, and from which completenucleotide sequences were determined. The complete nucleotide sequenceencodes a novel and highly divergent member of the FGF superfamily, andwas designated fibroblast growth factor homologous factor-1 (FHF-1). Thededuced amino acid sequence of FHF-1 contains 244 amino acids and is 27%identical to FGF-9, which is the member of the FGF family that sharesthe most homology with FHF-1. The nucleotide and deduced amino acidsequences of FHF-1 are shown in FIG. 5.

The FHF-1 sequence was used to search the National Center forBiotechnology Information (NCBI) database of expressed sequence tags(ESTs) and sequence tagged sites (STSs). One EST entry (DBEST ID 06895),derived from a human infant brain cDNA library (Adams, et al., NatureGenet. 4, 373-380, 1993) encoded a segment of 77 amino acids havingsignificant homology to the carboxyl-terminus of FHF-1, but nosignificant homology to other members of the FGF family. The polypeptidecontaining this amino acid segment was designated FHF-2. One STS entry(DBEST ID 76387; Brody, et al., Genomics 25, 238-247, 1995), derivedfrom human genomic DNA contained a putative exon encoding a proteinsegment with a high degree of homology to FHF-1 and a low degree ofhomology to other FGF family members. The polypeptide containing thisamino acid segment was designated FHF-3. Full-length FHF-2 and FHF-3cDNA clones were isolated from an adult human retina cDNA library, andwere found to encode proteins of 245 and 225 amino acids, respectively,each having greater than 58% amino acid identity to FHF-1 and to eachother. The nucleotide and deduced amino acid sequences of FHF-2 andFHF-3 are shown in FIG. 6 and FIG. 7, respectively.

A comparison of the sequences of FHF-1, FHF-2, and FHF-3 revealedseveral regions of high amino acid sequence conservation, and two ofthese regions were used to design degenerate oligonucleotide primers foruse in PCR. The primers have sequences that correspond to codons for theconserved amino acids at their 3′ ends (at residues 151-157 and 184-190in the FHF-1 sequence (SEQ ID NO:1) see FIG. 5, and below), andrestriction enzyme cleavage sites at their 5′ ends, to facilitatecloning of the resulting PCR products. A full degenerate sense strandprimer with a flanking EcoRI restriction site was synthesized for theamino acid sequence VFENYYV (SEQ ID NO:27; amino acids 151-157)(5′-CCGATCGAATTCGTNTT(T/C)GA(A/G)AA(T/C)TA(T/C)TA(T/C)GT-3′; SEQ IDNO:34). Three partially degenerate antisense primers with a flankingBamHI site were synthesized to include all possible codons for the aminoacid sequence MKGN(H/R)VK (SEQ ID NO:30; amino acids 184-190)

(5′-GCGATCGGATCCTTNAC(A/G)TG(A/G)TTNCC(T/C)TTCAT-3′ (SEQ ID NO:35);

5′-GCGATCGGATCCTTNAC(T/C)CT(A/G)TTNCC(T/C)TTCAT-3′ (SEQ ID NO:36); and

5′-GCGATCGGATCCTTNACNCG(A/G)TTNCC(T/C)TTCAT-3′ (SEQ ID NO:37)). Thethree pairs of sense and anti-sense primers were used in PCR reactionscontaining human, murine, and bovine genomic DNA templates and Thermusaquatics DNA polymerase and the reactions were carried out under thefollowing conditions: 1×(94° C., 7 minutes), 35×(45° C., 2 minutes; 72°C., 0.5 94° C., 0.5 minutes; 95° C., 0.25 minutes). PCR products werecleaved with EcoRi and BamHI, fractionated by preparative agarose gelelectrophoresis, subcloned into pBluescript, and individually sequenced.Analysis of the amplification products obtained using mouse, human, andbovine genomic DNA templates revealed that, in each of these species,this region of FHF-1, FHF-2, and FHF-3 is encoded within a single exon.This analysis also revealed a fourth class of FHF-like PCR productswhich was present in all three species and was found to encode anFHF-like protein, designated FHF-4. The PCR product corresponding toFHF-4 was used as a probe to isolate full-length cDNA clones from ahuman retina cDNA library and a developing mouse eye cDNA library. Aprotein of 247 amino acids having greater than 60% amino acid identityto FHF-1, FHF-2, and FHF-3, is predicted to be encoded by these clones.The nucleotide and deduced amino acid sequence of FHF-4 is shown in FIG.8.

FIG. 3 shows an alignment of the amino acid sequences of identified FHFsand previously characterized and published FGFs (1-9) and FIG. 4 is adendrogram of the identified FHFs and all the published FGF sequences.Pairwise comparisons between each FHF and the nine FGF family membersshow less than 30% amino acid sequence identity, while all pairwisecomparisons among the four FHFs show between 58% and 71% amino acidsequence identity. Between mouse and human orthologues, there is greaterthan 97% amino acid sequence identity. The murine FHFs differ from theirhuman orthologues by the amino acid substitutions listed below. Thefirst and last letters indicate the amino acids in the human and mousesequences, respectively, and the number indicates the position along thepolypeptide chain: FHF-1: Q86E; FHF-2: A2T, L136H; FHF-3: A58T, P60Q,Q180R, L197V, Q207R, A217T, P225H; and FHF-4: C40F, A181V, P221A, S244C.Thus, the four FHFs define a distinct and highly conserved branch of theFGF family.

FHFs 1-4 each lack a recognizable amino-terminal signal sequence. Amongthe nine previously characterized members of the FGF family, FGF-1,FGF-2, and FGF-9 are also distinguished by lacking a recognizableamino-terminal signal sequence (Abraham, et al., Science 233, 545-548,1986; Jaye, et al., Science 233, 541-545, 1986; Miyamoto, et al., MolCell Biol. 13, 4251-4259, 1993). Current evidence indicates that FGF-1and FGF-2 are synthesized in the cytosol and are released by a mechanismindependent of the ER-Golgi secretory pathway (Florkiewicz, et al., J.Cell Physiol. 162, 388-399, 1995; Jackson, et al., J. Biol. Chem. 270,33-36, 1995). Although FGF-9 lacks a cleavable amino-terminal signalsequence, it is glycosylated and is efficiently secreted from culturedglioma, Chinese hamster ovary, and COS cells, presumably via theER-Golgi pathway (Miyamoto, et al., Mol. Cell Biol. 13, 4251-4259,1993).

EXAMPLE 2 DEDUCED AMINO ACID SEQUENCE OF FHF-4

FIG. 8 shows the nucleotide and deduced amino acid sequences of humanFHF-4, which was derived from the human retina cDNA clones describedabove. As is mentioned above, the primary translation product of thehuman FHF-4 gene is predicted to be 247 amino acids in length. The humanFHF-4 initiator methionine codon shown in FIG. 8 at nucleotide positions78-80 and is the first in frame ATG; a good consensus ribosome bindingsite (CCACCATGG; Kozak, Nucleic Acids Res., 15:8125, 1987) is found atthis position. This choice of ATG conforms to the sites of translationinitiation in FHF-1, FHF-2, and FHF-3, as shown in the alignment in FIG.1. The next methionine codon in the FHF-4 open reading frame is located85 codons 3′ to the putative initiator methionine codon. Similar toFGF-1 and FGF-2, as well as FHFs 1-3, FHF-4 lacks a discemableamino-terminal signal sequence. Human FHF-4 has a single Asn-Lys-Sermotif at amino acids 242-244, which conforms to the consensus sequencefor asparagine-linked glycosylation, but this site is not conserved inthe highly homologous mouse FHF-4 sequence (see FIG. 3), suggesting thatit may not be used for glycosylation.

EXAMPLE 3 CHROMOSOMAL LOCALIZATION OF FHF GENES

In humans, FHF-1, FHF-2, FHF-3, and FHF-4 are located on chromosomes 3,X, 17, and 13, respectively. The chromosomal locations of FHF-1, FHF-2,and FHF-4, were determined by Southern blot hybridization of genomic DNAfrom rodent-human hybrid cell lines carrying individual humanchromosomes. For example, in the case of human FHF-4, a Southern blotcontaining restriction enzyme-digested DNA from a panel of 24human-mouse and human-hamster cell line, each containing a differenthuman chromosome (Oncor, Gaithersburg, Md.). Hybridization of a humanFHF-4 probe to human, mouse, and hamster genomic DNA produced distincthybridizing fragment sizes. Among the hybrid panels, the human-specifichybridization pattern was seen in the lanes corresponding to the hybridcell line carrying human chromosomes 1 and 13, suggesting that one ofthese cell lines contains additional genomic sequences derived from thechromosome present in the other cell line. To determine which of thesetwo human chromosomes contained the FHF-4 gene, the location of themouse gene was determined by interspecific backcross mapping. Asdiscussed further below, this analysis located the FHF-4 gene on mousechromosome 14, less than 1 cM from the Rap2a gene, and within a regionthat is syntenic with human chromosome 13q34. Taken together, these datashow that in humans, the FHF-4 gene maps to chromosome 13. The FHF-3locus is on human chromosome 17, as the STS described above thatencompasses one exon of FHF-3 was derived from human chromosome 17 andmaps near the BRCA-1 gene (Brody, et al., Genomics 25, 238-247, 1995).

The chromosomal locations of FHF-1, FHF-2, and FHF-4 in the mouse weredetermined using an inter-specific backcross mapping panel from crossesof (C57BL/6J×Mus spretus), F1×C57BL/6J. This mapping panel has beentyped for over 2100 loci, which are well distributed over all 19 mouseautosomes and the X-chromosome (Copeland and Jenkins, Trends Genet. 7,113-118, 1991). C57BL/6J and M. spretus DNAs were digested with severalrestriction enzymes and analyzed by Southern blot hybridization forinformative RFLPs. The chromosomal location of each locus was determinedby comparing its strain distribution in the backcross mice with thestrain distribution patterns for all other loci already mapped in thebackcross (FIG. 9). FHF-1 mapped to the proximal region of mousechromosome 16, 1.6 cM distal to Smst and 5.1 cM proximal to Apod. FHF-2mapped to the X chromosome, and did not recombine with Cd401 in 168 micetyped in common, suggesting that the two loci are within 1.8 cM of eachother (upper 95% confidence interval). As is mentioned above, FHF-4mapped to the distal region of chromosome 14, and did not recombine withRap2a in 142 mice typed in common, suggesting that the two loci arewithin 2.1 cM of each other. The FHF-3 gene was not mapped with thebackcross panel, as it did not reveal an informative RFLP when testedwith 14 restriction enzymes. The proximity of the human FHF-3 gene toBRCA-1 suggests that the mouse FHF-3 gene resides on chromosome 11 inthe region that is syntenic with the BRCA-1 region of human chromosome17. The FHF genes map in regions of the composite mouse linkage map(Mouse Genome Database, Jackson Laboratory, Bar Harbor, Me.), whichcontains a number of mutations that may be candidate FHF alleles.

EXAMPLE 4 TISSUE AND SUBCELLULAR DISTRIBUTIONS OF FHF RNAS

Expression Patterns of FHFs in the Mouse. The tissue distributions oftranscripts derived from each of the four identified FHF genes weredetermined in RNAse protection experiments. Analysis of RNA preparedfrom brain, eye, heart, kidney, liver, lung, spleen, and testis revealedthat each FHF is expressed in the brain, and FHF-1, FHF-2, and FHF-3 areexpressed in the eye, FHF-1 and FHF-4 are expressed in the testis, andFHF-2 is expressed in the heart (FIG. 10). In the brain, FHF-2transcripts are at least five-fold more abundant than the transcriptsfrom any of the other FHFs.

The patterns of FHF gene expression during development were studied byin situ hybridization experiments using sections obtained on gestationalday 11 (e11), gestational day 17 (e17), postnatal day 1 (P1), and fromadults. FHF-2 transcripts are abundant at each of the time pointsexamined, and are present in all divisions of the central and peripheralnervous systems, including the enteric nervous system (FIGS. 11A, 11C,11D, and 11F). Consistent with the RNAse protection experiments, FHF-2transcripts were observed in the developing heart at e17 (FIG. 11D).FHF-1, FHF-3, and FHF-4 transcripts were also observed to be widelydistributed throughout the developing nervous system (FIGS. 11B and11E). For example, at P1, FHF-1 was found to be highly expressed in theretina, olfactory epithelium, and olfactory bulb (FIG. 11E). At e11,FHF-1 and FHF-3 were also found in a segmental pattern in the body wall(FIG. 11B). In the adult brain, each FHF showed a distinct pattern ofexpression: FHF-1 transcripts were present at high levels in theolfactory bulb, and at lower levels in the cerebellum, the deepcerebellar nuclei, throughout the cortex, and in multiple midbrainstructures (FIG. 11G); FHF-2 transcripts were most abundant in thehippocampus and were present at lower levels in multiple brain areas(FIGS. 11H and 11I); FHF-3 transcripts were present in the olfactorybulb, hippocampus, and cerebellum, where they were most concentrated inthe Purkinje cell layer (FIG. 11J); and FHF-4 transcripts are present athigh levels throughout the granular layer of the cerebellum, and atlower levels in the hippocampus and olfactory bulb (FIGS. 11K and 11L).

Distribution of FHF-1 Immunoreactivity in Monkey Brain. The distributionof FHF-1 immunoreactivity in adult rhesus monkey brain was examinedusing affinity purified polyclonal anti-FHF-1 antibodies. Theseantibodies bind to recombinant FHF-1, but do not bind to recombinantFHF-2, as was determined by immunostaining of transfected 293 cells andby Western blotting. Although immunoreactivity with these antibodies isreferred to as ‘FHF-1 immunoreactivity’ the possibility exists thatother members of the FHF family are in part responsible for the observedimmunostaining.

FHF-1 immunoreactive somata are present throughout the rhesus monkeycerebral cortex, but they are unevenly distributed across layers in anyone area and display marked variations in density and distributionacross functional areas (FIGS. 12A-12E). The low magnificationphotomicrographs in FIG. 12 show that in primary visual, somatosensory,and auditory areas, a relatively high density of immunostained cells ispresent and that these cells occupy predominantly the middle layers. Incontrast, fewer and more widely scattered immunostained neurons arepresent in the precentral motor area and in the association cortex ofthe superior parietal lobule.

Common to each of these cortical areas is the presence of several FHF-1immunoreactive populations, the most prominent of which have relativelylarge (12-14 μm diameter), intensely immunoreactive somata. Otherneurons with smaller (8-10 μm diameter) and more lightly immunostainedsomata are also present in all areas (FIG. 12F). Co-localizationexperiments demonstrated that the FHF-1 immunoreactive neurons in thecerebral cortex make up a subpopulation of neurons that areimmunoreactive for the calcium-binding protein, parvalbumin (FIGS. 12Gand 12H), which have previously been shown to make up a subset ofgabanergic interneurons in the monkey cerebral cortex (Hendry, et al.,Exp. Brain Res. 76, 467-472, 1989). Variations in immunostained celldensity were also seen in the hippocampal formation, where intenselyimmunoreactive somata were relatively common in the subicular complex,but were widely scattered in the CA fields, the dentate hilus, and thedentate gyrus (FIG. 12I).

A different pattern of FHF-1 immunostaining was seen in the dorsalthalamus (FIG. 12J). Only a few of the many nuclei in this regioncontained immunoreactive neurons; most prominent among them was theprincipal somatosensory relay nucleus (the caudal ventroposterolateralnucleus, VPLC) and the visual relay nucleus (the lateral geniculatenucleus, LGN). In the LGN, both magnocellular and parvicellular layersare equally immunostained. FIG. 12J shows that in the LGN, ipsilateralto an eye deprived by occlusion since birth, FHF-1 immunostaining wasmarkedly lower in layers innervated by the deprived eye than in layersinnervated by the normal eye.

The relatively large sizes of the FHF-1 immunostained somata in nucleiof the dorsal thalamus suggests they are cell bodies of neurons thatsend their axons to the cerebral cortex. Localization of FHF-1immunoreactivity in neurons of VPLc and LGN that are lightlyimmunoreactive for parvalbumin supports this conclusion, since theseparvalbumin immunostained neurons in dorsal thalamus have been shown tobe thalamocortical neurons (Jones and Hendry, Eur. J. Neurosci. 1,222-246, 1989).

Outside of the dorsal thalamus, FHF-1 immunoreactive neurons werepresent in a diverse collection of subcortical nuclei, including theglobus pallidus and putamen, red nucleus, substantia nigra, and thirdnerve complex (FIG. 12K). In addition, large cells in the deep layers ofboth the superior and inferior colliculi were immunostained, as wereneurons in the deep cerebellar nuclei. In conclusion, FHF-1immunoreactivity was broadly distributed across the neuraxis, but ateach level it was present in subsets of neurons.

Subcellular Localization of FHF-1 in Transfected Cells. As noted above,of the nine FGFs described prior to this report, FGF-1 and FGF-2 aredistinguished by lacking of an amino terminal signal sequence and bysecretion via a pathway that is independent of the

ER and Golgi apparatus (Florkiewicz, et al., J. Cell Physiol. 162,388-399, 1995; Jackson, et al., J. Biol. Chem. 270, 33-36, 1995).Moreover, FGF-1 and a subset of FGF-2 isoforms, produced by alternativetranslation initiation, have been shown to accumulate in the nuclei ofthe cells in which they are synthesized, as well as in the nuclei oftarget cells (Imamaura, et al., Science 249, 1567-1570, 1990; Imamaura,et al., J. Biol. Chem. 267, 5676-5679, 1992; Bugler, et al., Molec. CellBiol, 573-577, 1991; Zhan, et al., Biochem. Biophys. Res. Comm. 188,982-991, 1992; Cao, et al., J. Cell Science 104, 77-87, 1993; Wiedlocha,et al., Cell 76, 1039-1051, 1994). These proteins contain a nuclearlocalization signal (NLS) that conforms closely to a consensus NLS. Thenuclear accumulation of FGF-1 and FGF-2 has raised the possibility thatthese proteins have modes of action in addition to those mediated bybinding and activating cell surface receptors.

Like FGF-1 and FGF-2, each of the four identified FHFs lacks a classicalsignal sequence and contains clusters of basic residues near its aminoterminus that could serve as an NLS. These observations suggest thatFHFs may resemble FGF-1 and FGF-2 in their mechanism of secretion and intheir subcellular localization. To test this possibility, the fate ofFHF-1 synthesized in transiently transfected 293 cells, which are humanembryonic kidney cells, was determined. In one set of experiments,biosynthetically labeled FHF-1 was found to accumulate to high levelsintracellularly, but was not detectably secreted into the medium (FIG.13). In contrast, human growth hormone produced in paralleltransfections was efficiently secreted (FIG. 13). As 293 cells cansecrete a wide variety of growth factors through the ER-Golgi pathway,this experiment is consistent with the proposal that FHFs are notsecreted via this route.

In a second set of experiments, immunostaining of transfected 293 cellsrevealed accumulation of FHF-1 in the nucleus, suggesting that theclusters of basic residues can function as an NLS. A similar result wasobtained with FHF-2. Two main classes of NLS motifs have been described,the classical and bipartite motifs (Boulikas, Crit. Rev. Eukaryotic GeneExpression 3, 193-227, 1993). The classical NLS contains a cluster ofsix lysine or arginine amino acids, and the bipartite NLS contains twoclusters of three or four basic amino acids separated by a ten aminoacid spacer. In FHF-1 the clusters of basic amino acids resemble moreclosely the bipartite NLS consensus. To identify and characterize theputative FHF-1 NLS, we determined the subcellular localization ofdeletion mutants of FHF-1 by immunostaining with anti-FHF-1 antibodies,and of fusions between FHF-1 and β-galactosidase by X-gal staining.These experiments show that the two clusters of arginines and lysines atamino acids 11-18 and 28-38 in FHF-1 make up the two basic regions of abipartite NLS. In the context of the FHF-1 protein, deletion of eithercluster produced a modest increase in the level of cytoplasmic FHF-1,but left significant nuclear accumulation (constructs 2 and 3; FIG. 14),while deletion of both regions abolished nuclear accumulation (construct4; FIGS. 14 and 15). To further characterize the minimal region requiredfor import, the first 56 or the first 69 residues of FHF-1 were fused tobeta-galactosidase and were shown to contain a functional NLS(constructs 5 and 6; FIGS. 14 and 15). Fusion of FHF-1 amino acids 1-22or 23-55 individually to beta-galactosidase failed to direct nuclearlocalization, indicating a requirement for both parts of the bipartiteNLS or a requirement for sequences distal to the first 55 residues. Withbeta-galactosidase fused to the first 69 amino acids of FHF-1, theresults of a deletion analysis resembled those seen with intact FHF-1(constructs 5, 9, 10, and 11; FIGS. 14 and 15). Finally, fusion of FHF-1amino acids 11-38, containing only the two clusters of basic amino acidsseparated by a 10 amino acid spacer, conferred nuclear localization,although less efficiently than the larger segments that contained thisregion, suggesting that amino acids 1-10 or 56-68, outside of thebipartite NLS consensus, play an ancillary role in nuclear localization.

It is to be understood that, while the invention has been described withreference to the above detailed description, the foregoing descriptionis intended to illustrate, but not to limit, the scope of the invention.Other aspects, advantages, and modifications of the invention are withinthe scope of the following claims. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

37 243 amino acids amino acid Not Relevant linear protein 1 Met Ala AlaAla Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg GluSer Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser 20 25 30 Pro SerLys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val 35 40 45 Phe SerLys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 Arg ProGlu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln 65 70 75 80 GlnGly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr 85 90 95 LysAsp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly 100 105 110Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala 115 120125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu 130135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser145 150 155 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp PheLeu Gly 165 170 175 Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg ValLys Lys Ile 180 185 190 Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile GluVal Cys Met Tyr 195 200 205 Arg Glu Pro Ser Leu His Glu Ile Gly Glu LysGln Gly Arg Ser Arg 210 215 220 Lys Ser Ser Gly Thr Pro Thr Met Asn GlyGly Lys Val Val Asn Gln 225 230 235 240 Asp Ser Thr 245 amino acidsamino acid Not Relevant linear protein 2 Met Ala Ala Ala Ile Ala Ser SerLeu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Arg Glu Lys Ser AsnAla Cys Lys Cys Val Ser Ser Pro Ser 20 25 30 Lys Gly Lys Thr Ser Cys AspLys Asn Lys Leu Asn Val Phe Ser Arg 35 40 45 Val Lys Leu Phe Gly Ser LysLys Arg Arg Arg Arg Arg Pro Glu Pro 50 55 60 Gln Leu Lys Gly Ile Val ThrLys Leu Tyr Ser Arg Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Ala AspGly Thr Ile Asp Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu PheAsn Leu Ile Pro Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly ValGln Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu TyrIle Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser ValPhe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met Ile Tyr 145 150 155 160 ArgGln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170 175Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala 180 185190 His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser 195200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr210 215 220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser MetSer 225 230 235 240 His Asn Glu Ser Thr 245 225 amino acids amino acidNot Relevant linear protein 3 Met Ala Ala Leu Ala Ser Ser Leu Ile ArgGln Lys Arg Glu Val Arg 1 5 10 15 Glu Pro Gly Gly Ser Arg Pro Val SerAla Gln Arg Arg Val Cys Pro 20 25 30 Arg Gly Thr Lys Ser Leu Cys Gln LysGln Leu Leu Ile Leu Ile Ser 35 40 45 Lys Val Arg Leu Cys Gly Gly Arg ProAla Arg Pro Asp Arg Gly Pro 50 55 60 Glu Pro Gln Leu Lys Gly Ile Val ThrLys Leu Phe Cys Arg Gln Gly 65 70 75 80 Phe Tyr Leu Gln Ala Asn Pro AspGly Thr Ile Asp Gly Thr Lys Asp 85 90 95 Glu Asn Ser Ser Phe Thr His PheAsn Leu Ile Pro Val Gly Leu Arg 100 105 110 Val Val Ile Ile Gln Ser AlaLys Leu Gly His Tyr Met Ala Met Asn 115 120 125 Ala Glu Gly Leu Asp TyrSer Ser Pro His Phe Thr Ala Glu Cys Arg 130 135 140 Phe Lys Glu Cys ValPhe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Ala 145 150 155 160 Leu Tyr ArgGln Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp 165 170 175 Lys GluGly Gln Val Met Lys Gly Asn Arg Val Lys Lys Ile Lys Ala 180 185 190 AlaAla His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu 195 200 205Pro Ser Leu His Ser Val Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala 210 215220 Pro 225 247 amino acids amino acid Not Relevant linear protein 4 MetAla Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser 20 25 30Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe 35 40 45Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln 50 55 60Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly 65 70 7580 Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp 85 9095 Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg 100105 110 Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn115 120 125 Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu CysLys 130 135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr SerSer Met 145 150 155 160 Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp PheLeu Gly Leu Asn 165 170 175 Lys Glu Gly Gln Ala Met Lys Gly Asn Arg ValLys Lys Ile Lys Pro 180 185 190 Ala Ala His Phe Leu Pro Lys Pro Leu GluVal Ala Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Asp Val Gly Glu ThrVal Pro Lys Pro Gly Val Thr 210 215 220 Pro Ser Lys Ser Thr Ser Ala SerAla Ile Met Asn Gly Gly Lys Pro 225 230 235 240 Val Asn Lys Ser Lys ThrThr 245 155 amino acids amino acid Not Relevant linear protein 5 Met AlaGlu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 AsnLeu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 AsnGly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 ThrArg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 SerVal Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 155amino acids amino acid Not Relevant linear protein 6 Met Ala Ala Gly SerIle Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly AlaPhe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys AsnGly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly ValArg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu GluArg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr LeuAla Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val Thr AspGlu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn ThrTyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 ArgThr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 208 amino acidsamino acid Not Relevant linear protein 7 Met Ala Pro Leu Gly Glu Val GlyAsn Tyr Phe Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val ProVal Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly GlnSer Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu AspHis Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr GlyPhe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly Thr ArgLys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile Ser Ile Ala ValGly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu GlyMet Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr GlnGlu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn ThrTyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 ArgTyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195200 205 243 amino acids amino acid Not Relevant linear protein 8 Met AlaAla Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 ArgGlu Ser Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser 20 25 30 ProSer Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val 35 40 45 PheSer Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 ArgPro Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln 65 70 75 80Gln Gly Tyr Phe Leu Glu Met His Pro Asp Gly Thr Ile Asp Gly Thr 85 90 95Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly 100 105110 Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala 115120 125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu130 135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile TyrSer 145 150 155 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala TrpGlu Leu Gly 165 170 175 Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn ArgVal Lys Lys Thr 180 185 190 Lys Pro Ser Ser His Phe Val Pro Lys Pro IleGlu Val Cys Met Tyr 195 200 205 Arg Glu Pro Ser Leu His Glu Ile Gly GluLys Gln Gly Arg Ser Arg 210 215 220 Lys Ser Ser Gly Thr Pro Thr Met AsnGly Gly Lys Val Val Asn Gln 225 230 235 240 Asp Ser Thr 245 amino acidsamino acid Not Relevant linear protein 9 Met Thr Ala Ala Ile Ala Ser SerLeu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Arg Glu Lys Ser AsnAla Cys Lys Cys Val Ser Ser Pro Ser 20 25 30 Lys Gly Lys Thr Ser Cys AspLys Asn Lys Leu Asn Val Phe Ser Arg 35 40 45 Val Lys Leu Phe Gly Ser LysLys Arg Arg Arg Arg Arg Pro Glu Pro 50 55 60 Gln Leu Lys Gly Ile Val ThrLys Leu Tyr Ser Arg Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Ala AspGly Thr Ile Asp Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu PheAsn Leu Ile Pro Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly ValGln Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu TyrThr Ser Glu His Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser ValPhe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155 160 ArgGln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170 175Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala 180 185190 His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser 195200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr210 215 220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser MetSer 225 230 235 240 His Asn Glu Ser Thr 245 225 amino acids amino acidNot Relevant linear protein 10 Met Ala Ala Leu Ala Ser Ser Leu Ile ArgGln Lys Arg Glu Val Arg 1 5 10 15 Glu Pro Gly Gly Ser Arg Pro Val SerAla Gln Arg Arg Val Cys Pro 20 25 30 Arg Gly Thr Lys Ser Leu Cys Gln LysGln Leu Leu Ile Leu Leu Ser 35 40 45 Lys Val Arg Leu Cys Gly Gly Arg ProThr Arg Gln Asp Arg Gly Pro 50 55 60 Glu Pro Gln Leu Lys Gly Ile Val ThrLys Leu Phe Cys Arg Gln Gly 65 70 75 80 Phe Tyr Leu Gln Ala Asn Pro AspGly Ser Ile Gln Gly Thr Pro Glu 85 90 95 Asp Thr Ser Ser Phe Thr His PheAsn Leu Ile Pro Val Gly Leu Arg 100 105 110 Val Val Thr Ile Gln Ser AlaLys Leu Gly His Tyr Met Ala Met Asn 115 120 125 Ala Glu Gly Leu Leu TyrSer Ser Pro His Phe Thr Ala Glu Cys Arg 130 135 140 Phe Lys Glu Cys ValPhe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Ala 145 150 155 160 Leu Tyr ArgGln Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp 165 170 175 Lys GluGly Arg Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Ala 180 185 190 AlaAla His Phe Val Pro Lys Leu Leu Glu Val Ala Met Tyr Arg Glu 195 200 205Pro Ser Leu His Ser Val Pro Glu Thr Ser Pro Ser Ser Pro Pro Ala 210 215220 His 225 247 amino acids amino acid Not Relevant linear protein 11Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala 1 5 1015 Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser 20 2530 Pro Ser Lys Asn Arg Gly Leu Phe Asn Gly Asn Leu Val Asp Ile Phe 35 4045 Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln 50 5560 Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly 65 7075 80 Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp 8590 95 Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg100 105 110 Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala MetAsn 115 120 125 Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro GluCys Lys 130 135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile TyrSer Ser Met 145 150 155 160 Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala TrpPhe Leu Gly Leu Asn 165 170 175 Lys Glu Gly Gln Val Met Lys Gly Asn ArgVal Lys Lys Thr Lys Pro 180 185 190 Ala Ala His Phe Leu Pro Lys Pro LeuGlu Val Ala Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Asp Val Gly GluThr Val Pro Lys Ala Gly Val Thr 210 215 220 Pro Ser Lys Ser Thr Ser AlaSer Ala Ile Met Asn Gly Gly Lys Pro 225 230 235 240 Val Asn Lys Cys LysThr Thr 245 245 amino acids amino acid Not Relevant linear protein 12Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Ser Trp 1 5 1015 Pro Thr Thr Gly Pro Gly Thr Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 2530 Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 4045 Tyr Cys Ala Thr Lys Tyr His Glu Gln Leu His Pro Ser Gly Arg Val 50 5560 Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 7075 80 Val Glu Val Gly Val Val Ala Ile Lys Gly Leu Phe Ser Gly Arg Tyr 8590 95 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Asp His Tyr Asn100 105 110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr AsnThr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Gly Ser Ser Gly Pro GlyAla Gln 130 135 140 Arg Gln Pro Gly Ala Gln Arg Pro Trp Tyr Val Ser ValAsn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg ArgThr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Gly His LysAsp His Glu Met Val Arg 180 185 190 Leu Leu Gln Ser Ser Gln Pro Arg AlaPro Gly Glu Gly Ser Gln Pro 195 200 205 Arg Gln Arg Arg Gln Lys Lys GlnSer Pro Gly Asp His Gly Lys Met 210 215 220 Glu Thr Leu Ser Thr Arg AlaThr Pro Ser Thr Gln Leu His Thr Gly 225 230 235 240 Gly Leu Ala Val Ala245 206 amino acids amino acid Not Relevant linear protein 13 Met SerGly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15 LeuAla Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30 ThrAla Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 40 45 SerLeu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 55 60 LysGlu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile 65 70 75 80Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Glu 85 90 95Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr Arg 100 105110 Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser Ile 115120 125 Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys130 135 140 Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Ile Phe Lys GluIle 145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr LysTyr Pro Gly 165 170 175 Met Glu Ile Ala Leu Ser Lys Asn Gly Lys Thr LysLys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe LeuPro Arg Leu 195 200 205 268 amino acids amino acid Not Relevant linearprotein 14 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu IleLeu 1 5 10 15 Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys GlyGln Pro 20 25 30 Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Asp Ser Ser SerArg Gln 35 40 45 Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser SerPro Ala 50 55 60 Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser SerPhe Gln 65 70 75 80 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr CysArg Val Gly 85 90 95 Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys ValAsn Gly Ser 100 105 110 His Glu Ala Asn Met Leu Ser Val Leu Glu Ile PheAla Val Ser Gln 115 120 125 Gly Ile Val Gly Ile Arg Gly Val Phe Ser AsnLys Phe Leu Ala Met 130 135 140 Ser Lys Lys Gly Lys Leu His Ala Ser AlaLys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe Gln GluAsn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175 Ala Ile His Arg Thr Glu LysThr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190 Asn Lys Pro Gly Lys AlaLys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gln His Ile Ser ThrHis Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln 210 215 220 Pro Glu Leu SerPhe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240 Ser ProIle Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255 AsnSer Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 198 amino acidsamino acid Not Relevant linear protein 15 Met Ser Arg Gly Ala Gly ArgLeu Gln Gly Thr Leu Trp Ala Leu Val 1 5 10 15 Phe Leu Gly Ile Leu ValGly Met Val Val Pro Ser Pro Ala Gly Thr 20 25 30 Arg Ala Asn Asn Thr LeuLeu Asp Ser Arg Gly Trp Gly Thr Leu Leu 35 40 45 Ser Arg Ser Arg Ala GlyLeu Ala Gly Glu Ile Ala Gly Val Asn Trp 50 55 60 Glu Ser Gly Tyr Leu ValGly Ile Lys Arg Gln Arg Arg Leu Tyr Cys 65 70 75 80 Asn Val Gly Ile GlyPhe His Glu Gln Val Leu Pro Asp Gly Arg Ile 85 90 95 Ser Gly Thr His GluGlu Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr 100 105 110 Val Glu Arg GlyVal Val Ser Leu Phe Gly Val Arg Ser Ala Leu Glu 115 120 125 Val Ala MetAsn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln 130 135 140 Glu GluCys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150 155 160Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr 165 170175 Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met Thr Val Thr 180185 190 His Phe Leu Pro Arg Ile 195 194 amino acids amino acid NotRelevant linear protein 16 Met His Lys Trp Ile Leu Thr Trp Ile Leu ProThr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val GlyThr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala ThrAsn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp TyrMet Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr GlnTrp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr GlnGlu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala ValGly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Leu Tyr AlaMet Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn GluAsp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn ThrTyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met PheVal Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys ThrLys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr215 amino acids amino acid Not Relevant linear protein 17 Met Gly SerPro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val LeuCys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30 Thr GlnHis Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40 45 Arg LeuIle Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60 Val GlnVal Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly 65 70 75 80 AspPro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95 ValArg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys 100 105 110Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120125 Phe Ile Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala 130135 140 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg145 150 155 160 Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His PheMet Lys 165 170 175 Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser LeuArg Phe Glu 180 185 190 Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu ArgGly Ser Gln Arg 195 200 205 Thr Trp Ala Pro Glu Pro Arg 210 215 1422base pairs nucleic acid single linear 18 GAATTCCGCA CACTGCGTTCGGGGTACCAA GTGGAAGGGG AAGAACGATG CCCAAAATAA 60 CAAGACGTGC CTGGGACCGCCCCGCCCCGC CCCCCGGCCG CCAGAGGTTG GGGAAGTTTA 120 CATCTGGATT TTCACACATTTTGTCGCCAC TGCCCAGACT TTGACTAACC TTGTGAGCGC 180 CGGGTTTTCG ATACTGCAGCCTCCTCAAAT TTTAGCACTG CCTCCCCGCG ACTGCCCTTT 240 CCCTGGCCGC CCAGGTCCTGCCCTCGCCCC GGCGGAGCGC AAGCCGGAGG GCGCAGTAGA 300 GGCTGGGGCC TGAGGCCCTCGCTGAGCAGC TATGGCTGCG GCGATAGCCA GCTCCTTGAT 360 CCGGCAGAAG CGGCAGGCGAGGGAGTCCAA CAGCGACCGA GTGTCGGCCT CCAAGCGCCG 420 CTCCAGCCCC AGCAAAGACGGGCGCTCCCT GTGCGAGAGG CACGTCCTCG GGGTGTTCAG 480 CAAAGTGCGC TTCTGCAGCGGCCGCAAGAG GCCGGTGAGG CGGAGACCAG AACCCCAGCT 540 CAAAGGGATT GTGACAAGGTTATTCAGCCA GCAGGGATAC TTCCTGCAGA TGCACCCAGA 600 TGGTACCATT GATGGGACCAAGGACGAAAA CAGCGACTAC ACTCTCTTCA ATCTAATTCC 660 CGTGGGCCTG CGTGTAGTGGCCATCCAAGG AGTGAAGGCT AGCCTCTATG TGGCCATGAA 720 TGGTGAAGGC TATCTCTACAGTTCAGATGT TTTCACTCCA GAATGCAAAT TCAAGGAATC 780 TGTGTTTGAA AACTACTATGTGATCTATTC TTCCACACTG TACCGCCAGC AAGAATCAGG 840 CCGAGCTTGG TTTCTGGGACTCAATAAAGA AGGTCAAATT ATGAAGGGGA ACAGAGTGAA 900 GAAAACCAAG CCCTCATCACATTTTGTACC GAAACCTATT GAAGTGTGTA TGTACAGAGA 960 ACCATCGCTA CATGAAATTGGAGAAAAACA AGGGCGTTCA AGGAAAAGTT CTGGAACACC 1020 AACCATGAAT GGAGGCAAAGTTGTGAATCA AGATTCAACA TAGCTGAGAA CTCTCCCCTT 1080 CTTCCCTCTC TCATCCCTTCCCCTTCCCTT CCTTCCCATT TACCCATTTC CTTCCAGTAA 1140 ATCCACCCAA GGAGAGGAAAATAAAATGAC AACGCAAGAC CTAGTGGCTA AGATTCTGCA 1200 CTCAAAATCT TCCTTTGTGTAGGACAAGAA AATTGAACCA AAGCTTGCTT GTTGCAATGT 1260 GGTAGAAAAT TCACGTGCACAAAGATTAGC ACACTTAAAA GCAAAGGAAA AAATAAATCA 1320 GAACTCCATA AATATTAAACTAAACTGTAT TGTTATTAGT AGAAGGCTAA TTGTAATGAA 1380 GACATTAATA AAGATGAAATAAACTTATTA CTTTCGGAAT TC 1422 1150 base pairs nucleic acid single linear19 AATTCCGCTT GCACAGTGTC CGCCGGGCGC AGGGGCCGAC CGCACGCAGT CGCGCAGTTC 60TGCCTCCGCC TGCCAGTCTC GCCCGCGATC CCGGCCCGGG GCTGTGGCGT CGACTCCGAC 120CCAGGCAGCC AGCAGCCCGC GCGGGAGCCG GACCGCCGCC GGAGGAGCTC GGACGGCATG 180CTGAGCCCCC TCCTTGGCTG AAGCCCGAGT GCGGAGAAGC CCGGGCAAAC GCAGGCTAAG 240GAGACCAAAG CGGCGAAGTC GCGAGACAGC GGACAAGCAG CGGAGGAGAA GGAGGAGGAG 300GCGAACCCAG AGAGGGGCAG CAAAAGAAGC GGTGGTGGTG GGCGTCGTGG CCATGGCGGC 360GGCTATCGCC AGCTCGCTCA TCCGTCAGAA GAGGCAAGCC CGCGAGCGCG AGAAATCCAA 420CGCCTGCAAG TGTGTCAGCA GCCCCAGCAA AGGCAAGACC AGCTGCGACA AAAACAAGTT 480AAATGTCTTT TCCCGGGTCA AACTCTTCGG CTCCAAGAAG AGGCGCAGAA GAAGACCAGA 540GCCTCAGCTT AAGGGTATAG TTACCAAGCT ATACAGCCGA CAAGGCTACC ACTTGCAGCT 600GCAGGCGGAT GGAACCATTG ATGGCACCAA AGATGAGGAC AGCACTTACA CTCTGTTTAA 660CCTCATCCCT GTGGGTCTGC GAGTGGTGGC TATCCAAGGA GTTCAAACCA AGCTGTACTT 720GGCAATGAAC AGTGAGGGAT ACTTGTACAC CTCGGAACTT TTCACACCTG AGTGCAAATT 780CAAAGAATCA GTGTTTGAAA ATTATTATGT GACATATTCA TCAATGATAT ACCGTCAGCA 840GCAGTCAGGC CGAGGGTGGT ATCTGGGTCT GAACAAAGAA GGAGAGATCA TGAAAGGCAA 900CCATGTGAAG AAGAACAAGC CTGCAGCTCA TTTTCTGCCT AAACCACTGA AAGTGGCCAT 960GTACAAGGAG CCATCACTGC ACGATCTCAC GGAGTTCTCC CGATCTGGAA GCGGGACCCC 1020AACCAAGAGC AGAAGTGTCT CTGGCGTGCT GAACGGAGGC AAATCCATGA GCCACAATGA 1080ATCAACGTAG CCAGTGAGGG CAAAAGAAGG GCTCTGTAAC AGAACCTTAC CTCCAGGTGC 1140TGTTGAATTC 1150 961 base pairs nucleic acid single linear 20 GAATTCCGGCTCTTGGGGAG CCCAGCGCGC TCCGGGCGCC TGCCGGTTTG GGGGTGTCTC 60 CTCCCGGGGCGCTATGGCGG CGCTGGCCAG TAGCCTGATC CGGCAGAAGC GGGAGGTCCG 120 CGAGCCCGGGGGCAGCCGGC CGGTGTCGGC GCAGCGGCGC GTGTGTCCCC GCGGCACCAA 180 GTCCCTTTGCCAGAAGCAGC TCCTCATCCT GCTGTCCAAG GTGCGACTGT GCGGGGGGCG 240 GCCCGCGCGGCCGGACCGCG GCCCGGAGCC TCAGCTCAAA GGCATCGTCA CCAAACTGTT 300 CTGCCGCCAGGGTTTCTACC TCCAGGCGAA TCCCGACGGA AGCATCCAGG GCACCCCAGA 360 GGATACCAGCTCCTTCACCC ACTTCAACCT GATCCCTGTG GGCCTCCGTG TGGTCACCAT 420 CCAGAGCGCCAAGCTGGGTC ACTACATGGC CATGAATGCT GAGGGACTGC TCTACAGTTC 480 GCCGCATTTCACAGCTGAGT GTCGCTTTAA GGAGTGTGTC TTTGAGAATT ACTACGTCCT 540 GTACGCCTCTGCTCTCTACC GCCAGCGTCG TTCTGGCCGG GCCTGGTACC TCGGCCTGGA 600 CAAGGAGGGCCAGGTCATGA AGGGAAACCG AGTTAAGAAG ACCAAGGCAG CTGCCCACTT 660 TCTGCCCAAGCTCCTGGAGG TGGCCATGTA CCAGGAGCCT TCTCTCCACA GTGTCCCCGA 720 GGCCTCCCCTTCCAGTCCCC CTGCCCCCTG AAATGTAGTC CCTGGACTGG AGGTTCCCTG 780 CACTCCCAGTGAGCCAGCCA CCACCACAAC CTGTCTCCCA GTCCTGCTCT CACCCCTGCT 840 GCCACACACATGCCCTGAGC AGCCAGGTCC CACTAGGTGC TCTACCCTGA GGGAGCCTAG 900 GGGCTGACTGTGACTTCCGA GGCTGCTGAG ACCCTTAGAT CTTTGGGCCT AGGAGGGAGT 960 C 961 971base pairs nucleic acid single linear 21 CGCCGCCTTC CCCTCCGGTGCCCCCGGCTC GCCGTCCTCC CGCGCCCTCC CTCCCCGGAC 60 CCGTTCCCGG GGCCACCATGGCCGCGGCCA TCGCTAGCGG CTTGATCCGC CAGAAGCGGC 120 AGGCGCGGGA GCAGCACTGGGACCGGCCGT CTGCCAGCAG GAGGCGGAGC AGCCCCAGCA 180 AGAACCGCGG GCTCTGCAACGGCAACCTGG TGGATATCTT CTCCAAAGTG CGCATCTTCG 240 GCCTCAAGAA GCGCAGGTTGCGGCGCCAAG ATCCCCAGCT CAAGGGTATA GTGACCAGGT 300 TATATTGCAG GCAAGGCTACTACTTGCAAA TGCACCCCGA TGGAGCTCTC GATGGAACCA 360 AGGATGACAG CACTAATTCTACACTCTTCA ACCTCATACC AGTGGGACTA CGTGTTGTTG 420 CCATCCAGGG AGTGAAAACAGGGTTGTATA TAGCCATGAA TGGAGAAGGT TACCTCTACC 480 CATCAGAACT TTTTACCCCTGAATGCAAGT TTAAAGAATC TGTTTTTGAA AATTATTATG 540 TAATCTACTC ATCCATGTTGTACAGACAAC AGGAATCTGG TAGAGCCTGG TTTTTGGGAT 600 TAAATAAGGA AGGGCAAGCTATGAAAGGGA ACAGAGTAAA GAAAACCAAA CCAGCAGCTC 660 ATTTTCTACC CAAGCCATTGGAAGTTGCCA TGTACCGAGA ACCATCTTTG CATGATGTTG 720 GGGAAACGGT CCCGAAGCCTGGGGTGACGC CAAGTAAAAG CACAAGTGCG TCTGCAATAA 780 TGAATGGAGG CAAACCAGTCAACAAGAGTA AGACAACATA GCCAGATCCT CACAGGTGTT 840 GTGACTTATT CGTCCTGAGCACAGTTGAGT GATTTATCCT CACCAGACAT TCCTGCTCCG 900 TGGCTGAAGA GCAGCAGGAAGTAAGCTAAT GCTTATTCTT TGCTGTCTCC GAACTTCTCT 960 GTTGCAAGTG G 971 14amino acids amino acid Not Relevant linear protein Xaa in position 4 isIsoleucine or Leucine; Xaa in position 7 is Serine or Glycine. 22 AlaAla Ala Xaa Ala Ser Xaa Ser Leu Ile Arg Gln Lys Arg 1 5 10 9 amino acidsamino acid Not Relevant linear protein Xaa in position 9 is Arginine orLysine. 23 Pro Gln Leu Lys Gly Ile Val Thr Xaa 1 5 13 amino acids aminoacid Not Relevant linear protein Xaa in position 2 is Leucine orHistidine. 24 Thr Xaa Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val 1 5 109 amino acids amino acid Not Relevant linear protein Xaa in position 4is Glycine, Serine, or Alanine; Xaa in position 7 is Tyrosine orLeucine. 25 Ala Met Asn Xaa Glu Gly Xaa Leu Tyr 1 5 10 amino acids aminoacid Not Relevant linear protein Xaa in position 3 is Serine orCysteine. 26 Lys Glu Xaa Val Phe Glu Asn Tyr Tyr Val 1 5 10 7 aminoacids amino acid Not Relevant linear protein 27 Val Phe Glu Asn Tyr TyrVal 1 5 9 amino acids amino acid Not Relevant linear protein Xaa inposition 4 is Alanine or Glycine; Xaa in position 6 is Phenylalanine orTyrosine. 28 Ser Gly Arg Xaa Trp Xaa Leu Gly Leu 1 5 10 amino acidsamino acid Not Relevant linear protein Xaa in position 5 is Arginine orHistidine; Xaa in position 9 is Threonine or Asparagine. 29 Met Lys GlyAsn Xaa Val Lys Lys Xaa Lys 1 5 10 7 amino acids amino acid Not Relevantlinear protein Xaa in position 5 is Histidine or Arginine. 30 Met LysGly Asn Xaa Val Lys 1 5 10 amino acids amino acid Not Relevant linearprotein Xaa in position 2 is Cysteine or Alanine; Xaa in position 5 isArginine, Glutamine, or Lysine. 31 Val Xaa Met Tyr Xaa Glu Pro Ser LeuHis 1 5 10 30 base pairs nucleic acid single linear 32 GACGAGATATTAGAATTCTA CTCGNNNNNN 30 33 base pairs nucleic acid single linear 33CCCCCCCCCG ACGAGATATT AGAATTCTAC TCG 33 32 base pairs nucleic acidsingle linear 34 CCGATCGAAT TCGTNTTYGA RAAYTAYTAY GT 32 32 base pairsnucleic acid single linear 35 GCGATCGGAT CCTTNACRTG RTTNCCYTTC AT 32 32base pairs nucleic acid single linear 36 GCGATCGGAT CCTTNACYCTRTTNCCYTTC AT 32 32 base pairs nucleic acid single linear 37 GCGATCGGATCCTTNACNCG RTTNCCYTTC AT 32

What is claimed is:
 1. A substantially pure fibroblast growth factorhomologous factor-4 (FHF-4) polypeptide, comprising SEQ ID NO:4.
 2. TheFHF-4 polypeptide of claim 1, further comprising a carrier protein. 3.The FHF-4 polypeptide of claim 2, wherein the carrier protein is keyholelimpet hemocyanin, thyroglobulin, bovine serum albumen, or tetanustoxoid.
 4. The FHF-4 polypeptide of claim 1, which is bound to a matrix.5. The FHF-4 polypeptide of claim 4, wherein the matrix is glass,polystyrene, polypropylene, polyethylene, dextran, amylase, cellulose,polyacrylamide, agarose or magnetite.
 6. The FHF-4 polypeptide of claim1, further comprising a detectable label.
 7. The FHF-4 polypeptide ofclaim 6, wherein the detectable label is an enzyme, a radioisotope, afluorescent compound, a colloidal metal, a chemiluminescent compound, aphosphorescent compound or a bioluminescent compound.
 8. A composition,comprising the FHF-4 polypeptide of claim 1 and an adjuvant.